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Home , Butaclamol, Chloroxine, Etazolate, Lasalocid, Motesanib, Naltriben

Identification of pathways that regulate circadian rhythms using a larval zebrafish small molecule screen

Eric A. Mosser1,†, Cindy N. Chiu1,#, T. Katherine Tamai2, Tsuyoshi Hirota3,4, Suna Li1, May Hui1,

Amy Wang1, Chanpreet Singh1, Andrew Giovanni5,^, Steve A. Kay6 and David A. Prober1,*

1Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA,

91125; 2Centre for Cell and Molecular Dynamics, Department of Cell and Developmental Biology,

University College London, London, United Kingdom, WC1E 6BT; 3PRESTO, JST, Nagoya, Japan,

464-8601; 4Institute of Transformative Bio-Molecules, Nagoya University, Nagoya, Japan, 464-

8601; 5Sunovion Pharmaceuticals, Marlborough, MA, 01752; 6Department of Neurology, Keck

School of Medicine, University of Southern California, Los Angeles, CA, 90089.

† Program in Biological Sciences, Northwestern University

# Department of Neurobiology, Northwestern University

^ Deceased

* Corresponding author: David A. Prober (email: [email protected]) Figure S1 3 2 2 0 1 2 3

S.D. - - - Figure S1. Clustergram summarizing results of the screen. All that affected at Amplitude Period Phase phase Δ Net least one of the measured Thiophene circadian parameters by at least 3 BX795 PDK1 inhibitor Nisodipine standard deviations from same- GW-7647 PPARalpha activator plate DMSO controls were PI3K gamma inhibitor PI3K gamma inhibitor D2 antagonist analyzed using hierarchical AS 252424 PI3K inhibitor clustering and are depicted in this L-type Ca channel inhibitor figure. The same clustergram is NSAID alpha antagonist shown without drug annotations in GABA-A activator Fig. 1D. nicotinic ACh Penitrem A inhibitor GC25 PCA4248 PAF antagonist Ethinyl GW-7647 PPARalpha activator 147 24 0 H1R antagonist SSRI UK418 SSRI Ca channel blocker Tyrphostin 8 calcineurin inhibitor Vinpocetin PDE inhibitor MAOI Suloctidil aggregation inhibitor CGP 13501 GABA-B activator L 703606 NK1 potassium channel blocker GW-7647 PPARalpha activator D2 receptor antagonist Tolcapone COMT inhibitor Acetyl farnesyl cysteine farnesylation inhibitor HCl hERG channel blocker reverse transcriptase inhibitor CyPPA potassium channel activator Chlorophyllide antineoplastic JFD00244 SIRT inhibitor Ivermectin nicotinic ACh receptor modulator Clinafoxacin Chloride mAch antagonist Hydroxycampothecin topoisomerase 1 inhibitor Chlorpheniramine maleate H1R antagonist D2 receptor antagonist NSAID Sertaline SSRI antibiolic antibacterial Deprenyl MAO-B inhibitor Daunorubicin antineoplastic WB 4101 alpha1A adrenoceptor inhibitor Anethole trithione increases response Candicidin CGP 7930 GABA-B activator Clothiapine receptor antagonist Nocodazole tubulin polymerization inhibitor SNRI Vindesine sulfate anti-mitotic HCl SSRI Roxithromycin antibiotic HCl D2 receptor antagonist Piperidolate HCl antispasmodic GC248 Pimozide antagonist Prozac SSRI Phenyl aminosalicylate Deprenyl MAO-B inhibitor Figure S1 (continued) 3 2 2 0 1 2 3

S.D. - - - Amplitude Period Phase phase Δ Net

Azelastine HCl H1R antagonist GC114 Mebendazole ACh esterase inhibitor antifungal PDE1 inhibitor Pyrethrins insecticide Vorinostat histone deacetylase inhibitor HCl PDE inhibitor Ethopropazine HCl inhibitor calcium channel blocker antagonist PAC 1 caspase 3 inhibitor Isotharine mesylate beta-andrenoceptor agonist Hydroflumethiazide antihypertensive Pfizerpen antibiotic IC 261 CK1 inhibitor Hexylresorcinol antihelmintic CGS 12066A 5-HT1B antagonist Gemfibrozil PPARalpha antagonist MAO inhibitor UK57 H1R antagonist SSRI Beta-estradiol estrogen prostaglandin antagonist Haloperidol D2 receptor antagonist AMG 9810 TRPV1 receptor antagonist K 185 receptor antagonist Dioxybenzone sunscreen NSAID Lyso C16 PAF platelet activating factor Mebendazole ACh esterase inhibitor Asarylaldehyde fly attractant L-type Ca channel inhibitor antimuscarinic Chlorpheniramine H1R antagonist kinase inhibitor Canrenone aldosterone antagonist Chenodiol synthesis inhibitor FKBP Oxaprozin NSAID Aminopyrine analgesic non-steroidal anti- dopamine receptor antagonist ALX 5407 transporter inhibitor calcium channel blocker L-type Ca channel inhibitor Paroxetine HCl SSRI Butaclamol dopamine receptor antagonist mACh antagonist SSRI NSAID dopamine receptor antagonist SNRI agonist antibiotic CAS 879127-07-8 EGFR inhibitor Vincristine sulfate microtubule assembly inhibitor Vinblastine microtubule assembly inhibitor Amsacrine topoisomerase inhibitor CCK receptor antagonist Psora-4 Kv1.3 channel inhibitor EGFR inhibitor NSAID Chlorphenyl dichloroethane antiadrenal S N acetylpenicillamine donor Figure S1 (continued) 3 2 2 0 1 2 3

S.D. - - - Amplitude Period Phase phase Δ Net

Cyclosporin immunosuppressant NSAID SC 51322 EP1 receptor antagonist SU-6656 Src kinase inhibitor NSAID Nimodipine L-type Ca channel inhibitor VEGFR inhibitor NSAID Denbufylline PDE inhibitor SC 19220 EP1 receptor antagonist SU-6656 Src kinase inhibitor Antibacterial Oxaprozin NSAID SU-6656 Src kinase inhibitor Meloxicam NSAID Rapamycin mTOR inhibitor WIN 62577 NK1 receptor antagonist Rapamycin mTOR inhibitor Ibuprofen NSAID Polymyxin B sulfate antibiotic PF562271 FAK inhibitor Dibucaine HCl anesthetic Motesanib inhibitor xinafoate beta2 agonist Rapamycin mTOR inhibitor NSAID Rocuronium AChR antagonist Ibuprofen NSAID Adapalene Estradiol estrogen Pravadoline NSAID Nalidixic acid antibiotic AV951 VEGFR inhibitor VEGFR inhibitor IV VEGFR inhibitor Tivozanib VEGFR inhibitor Chloroxine antibiotic AC220 FLT3 inhibitor Cdk1/2 inhibitor III Cdk1/2 inhibitor anticholenergic Nonoxynol 9 spermatocide Indirubin-3-monoxime GSK3-beta inhibitor Pyrantel pamoate neuromuscular blocking agent sulfate GABA-A antagonist NSAID Mitomycin C antibiotic sirtuin activator tyrosine kinase inhibitor NSAID Tranilast PGE2 inhibitor CP-868388 PPARalpha agonist CP-868388 PPARalpha agonist CP-868388 PPARalpha agonist alpha- 5 Nitro-2-3-phenylpropyl benzoic acid Cl channel blocker receptor antagonist Merbromin antibacterial Fendendazole antihelmintic Daunorubicin antineoplastic Pyrvinium pamoate antihelmintic Resveratrol sirtuin activator Naproxen NSAID diuretic succinate 5HT receptor agonist Pararosaniline antihelmintic Rosiglitazone PPAR agonist Cis Piperidine NMDAR agonist estrogen receptor agonist Figure S1 (continued) 3 2 2 0 1 2 3

S.D. - - - Amplitude Period Phase phase Δ Net

Proflavine bacteriostatic Chlorocresol methylphenol NSAID Valganciclovir antiviral reverse transcription inhibitor Estradiol acetate estrogen HBr beta2-adrenoreceptor agonist 3-tropanyl-3-5-dichlorobenzoate 5-HT3 receptor antagonist Na channel agonist glucocorticoid Flupenthixol dopamine receptor antagonist Oxolamine citrate cough suppressant UK22 5-alpha-androstan androgen Baccatin III precursor to Lomifylline PDE inhibitor Cardene Ca channel blocker Eicosadienoic acid lipoxygenase inhibitor PP1 PP2A inhibitor dopamine receptor agonist 5-HT2 receptor antagonist Temefos insecticide Aklavine HCl antibacterial Amlexanox anti-inflammatory PKC inhibitor HCl 5HT1A receptor agonist Hydrocortisone glucocorticoid Finasteride type II 5a reductase inhibitor U 50488 k- agonist N-Phenylanthranilic misc. channels R Desmethyldeprenyl MAO-B inhibitor Selamectin antiparasitic Beclomethasone inhibitor UK420 Chlormadinone steroidal progestin Resveratrol sirtuin activator Metformin AMPK activator Resveratrol sirtuin activator 17 Allylamino geldanamycin HSP-90 inhibitor Resveratrol sirtuin activator Atropine methyl Br mACh receptor antagonist SNS 032 CDK inhibitor PP2 Src kinase inhibitor hydrocortisone glucocorticoid Halometasone Isonicotinamide sirtuin activator phenethyl ester NFkB inhibitor Resveratrol sirtuin activator Resveratrol sirtuin activator corticosteroid vasodilator L-type Ca channel inhibitor Melengestrol steroidal progestin Warfarin vitamin K reductase inhibitor Cortisone acetate corticosteroid MAPK inhibitor Dicumarol anticoagulant Oleic acid PKC activator glucocorticoid Metformin AMPK activator AICAR AMPK activator corticosteroid TMB HCl calcium antagonist SB415286 GSK3 inhibitor Adapalene retinoid AICAR AMPK activator SU-6656 Src kinase inhibitor Figure S1 (continued) 3 2 2 0 1 2 3

S.D. - - - Amplitude Period Phase phase Δ Net

Capsaicin TRPV1 activator BX795 PDK1 inhibitor Clofibrate lipoprotein lipase activator PIM1/2 kinase inhibitor IV PIM1/2 kinase inhibitor 50 22 6 glucocorticoid Metformin AMPK activator Dexamethasone glucocorticoid SB 239063 p38 MAPK inhibitor Roscovitine CDK inhibitor UK343 glucocorticoid Goat’s rue extract CCK receptor antagonist K channel blocker AChE inhibitor VMAT inhibitor Resveratrol sirtuin activator CGP 74514A CDK1 inhibitor Resveratrol sirtuin activator TG003 Cdc2-like kinase inhibitor AChE inhibitor glucocorticoid UK326 glucocorticoid Isonicotinamide sirtuin activator Acetylsalicylic acid NSAID TOVOK HER2 and EGFR antagonist H 89 PKA inhibitor Natriben methanesulfonate delta2 opiod receptor antagonist Resveratrol sirtuin activator Resveratrol sirtuin activator Rhodblock 6 Rho kinase inhibitor GNE 490 PI3K inhibitor Oxibendazole antihelmintic antagonist Arecaidine mACh receptor agonist Phenindione anticoagulant Amifostine radioprotective agent Phloretin L-type Ca channel blocker AS 252424 PI3K gamma inhibitor Ro 31 8220 PKC inhibitor Aztreonam cell wall synthesis inhibitor tyrosine kinase inihbitor KU0063794 mTORC1/2 inhibitor PF04217903 C-Met inihibitor SCH 51344 Ras/Rac transformation blocker alpha-2 adrenoreceptor inhibitor estrogen receptor modulator AG 9 EGFR kinase inhibitor Climbazole antifungal Cerulenin biosynthesis inhibitor Indirubin 3 monoxime GSK-3b inhibitor alpha-1 adrenoceptor Enalaprilat ACEblocker inhibitor Deflazacort glucocorticoid H2R antagonist RU 24969 5HTA1/A2 receptor agonist antidepressant M2 mACh receptor antagonist Metformin AMPK activator SSR112050 CK1 inhibitor SSR112050 CK1 inhibitor SSR112050 CK1 inhibitor SSR112050 CK1 inhibitor N9 Isopropylolomoucine CDC-2 kinase inhibitor non- Roscovitine CDK inhibitor TAK 715 P38 MAPK inhibitor Roscovitine CDK inhibitor corticosteroid Figure S1 (continued) 3 2 2 0 1 2 3

S.D. - - - Amplitude Period Phase phase Δ Net

ABT 737 Bcl-2 inhibitor VEGF receptor TK inhibitor II VEGFR inhibitor MEK1/2 inhibitor II MEK1/2 inhibitor GW-7647 PPARalpha activator H1R antagonist BI 2536 R PLK inhibitor BX912 PDK1 inhibitor Gefitinib EGFR inhibitor Bohemine CDK1 inhibitor EGFR inhibitor Isonicotinamide sirtuin activator Isonicotinamide sirtuin activator Isonicotinamide sirtuin activator Isonicotinamide sirtuin activator NO synthetase inhibitor Figure S2

A CK1 inhibitor B AMPK activator MAPK inhibitor antibiotic DMSO CDK inhibitor kinase inhibitor 15 26 RTK inhibitor PI3K inhibitor Metformin Amplitude Period Phase phase Δ Net 10 * Roscovitine CDK inhibitor 5 25 VEGF receptor TK inhibitor II VEGFR inhibitor 0 Zafirlukast leukotriene receptor antagonist -5 24 Oxibendazole anti-helminthic -10 Arecaidine muscarinic receptor agonist Period (h) -15 Vandetanib EGFR inhibitor

Luminescence (a.u.)Luminescence -20 23 Bohemine CDK inhibitor 24 48 72 96 DMSO 1 mM Gefitinib EGFR inhibitor Time (h) Metformin Tracazolate modulates GABA receptor Nortriptyline HCl Enalaprilat ACE inhibitor C Sirtuin activator Ranitidine H2R inhibitor DMSO Deflazacort glucocorticoid 10 Isonicotinamide 27 *** Isonicotinamide sirtuin activator

Isonicotinamide sirtuin activator 5 ) 26 h Acetylsalicylic Acid NSAID 300uM Resveratrol sirtuin activator 0 25 100uM Resveratrol sirtuin activator

Naltriben methanesulfonate antagonist -5 Period ( 24 Tracazolate modulates GABA -10 23 N9 Isopropylolomucine receptor (a.u.)Luminescence CDK inhibitor 24 48 72 96 DMSO 2 μM Rhodblock 6 Rho kinase inhibitor Time (h) Isonicotinamide GNE 490 PI3K inhibitor PF04217903 MET inhibitor Arecaidine muscarinic receptor agonist D PI3K inhibitor H 89 PKA inhibitor DMSO Phloretin 8 27 L-type Ca channel inhibitor LY-294002 RU 24969 5HT receptor agonist * 4 26 Ranitidine H2R inhibitor ) h AS 252424 PI3K inhibitor 0 25 Ro 31 8220 PKC inhibitor BX912 PDK1 inhibitor -4 24 Bosutinib tyrosine kinase inhibitor Period ( BI 2536 R PLK1 inhibitor -8 23 Cerulenin antifungal (a.u.)Luminescence 24 48 72 96 DMSO 25 μM Climbazole antifungal Time (h) LY-294002 Raloxifene estrogen receptor modulator Yohimbine alpha-2 adrenoreceptor inhibitor AG 9 E CDK inhibitor Aztreonam antibiotic (gram negative) DMSO Raloxifene estrogen receptor modulator 8 Roscovitine 28 Indirubin-3-monoxime GSK3-beta inhibitor 4 27 **

ABT 737 Bcl-2 inhibitor ) h SCH 51344 Ras/Rac transformation blocker 26 0 Dehydroepiandrosterone corticosteroid Phloretin L-type Ca channel inhibitor 25 -4 Amifostine radio protective agent Period ( 24 Nortriptyline HCl tricyclic antidepressant -8 23 Arecaidine muscarinic receptor agonist (a.u.)Luminescence 24 48 72 96 DMSO 30 μM SSR112050 CK1 inhibitor Time (h) Roscovitine SSR112050 CK1 inhibitor SSR112050 CK1 inhibitor SSR112050 CK1 inhibitor F 34 * Roscovitine CDK inhibitor TAK 715 P38 MAPK inhibitor 32 Roscovitine CDK inhibitor 30 N9 Isopropylolomucine CDK inhibitor SSR112050 CK1 inhibitor 28 Raloxifene estrogen receptor modulator

Period (h) 26 Yohimbine alpha-2 adrenoreceptor inhibitor SCH 51344 Ras/Rac transformation blocker 24

22 Cortisone acetate glucocorticoid DMSO 0.6250.625 μM 1.251.25 μM 2.52.5 μM 5.05 μM Prednisolone glucocorticoid Dicumarol anticoagulant [A002195858] Oleic acid PKC activator Dexamethasone glucocorticoid Metformin AMPK activator AICAR AMPK activator Corticosterone corticosteroid TMB HCl calcium antagonist SB415286 GSK3 inhibitor S.D. Adapalene retinoid 3 2 2 0 1 2 3

AICAR AMPK activator - - - SU-6656 Src kinase inhibitor Figure S2. Drugs that increased Tg(per3:luc) luminescence circadian period length. (A) Clusters of drugs that increased period length by at least 3 standard deviations from the mean of same-plate DMSO controls is shown. Color-coding indicates drug classes. Compounds that increased period length include AMPK activator metformin (B), sirtuin activator isonicotinamide (C), PI3K inhibitor LY-204002 (D) and CDK inhibitor roscovitine (E). Line graphs show mean and bar graphs show mean ± SEM for 8 animals. *P<0.05, **P<0.01, ***P<0.001 by ANOVA with Tukey’s test. (F) Treatment of Rat-1 fibroblasts stably transfected with a mPer1-luc reporter construct with the CK1 inhibitor A002195858 resulted in significantly increased period length at 5 μM. Mean ± SEM for triplicate samples are shown. *P<0.05 by Kruskal-Wallis test corrected for multiple comparisons to DMSO using the Dunn test. A CK1 inhibitor DMSO B CK1 inhibitor DMSO 6 SSR112050 34 * 7 SSR112050 32 * 6 5 32 30 per3

pe1b 5 4 30 4 28 3 28 3 26 2 26 2 Period (h) Period (h) 24 1 24 1 normalized normalized expression (a.u.) normalized expression (a.u.) 0 22 0 6am 6am 22 12pm 6pm 12am 6am 12pm 6pm 12am 6am 12pm DMSO 30 μM 12pm 6pm 12am 12pm 6pm 12am 12pm DMSO 30 μM SSR112050 SSR112050

C Anti-inflammatory: NSAID D Anti-inflammatory: NSAID DMSO 20 DMSO 3.0 14 2.0 * Naproxen Naproxen 2.5 12 * 15 1.5 per3 10 2.0 per1b 8 10 1.5 1.0 6 1.0 5 4 0.5 0.5 2 normalized normalized expression (a.u.) Amplitude (RLU) expression (a.u.) Amplitude (RLU) normalized normalized 0 0.0 0 0.0 12pm 6pm 12am 6am 12pm 6pm 12am 6am 12pm 6pm 12am DMSO 25 μM 12pm 6pm 12am 6am 12pm 6pm 12am 6am 12pm 6pm 12am DMSO 25 μM Naproxen Naproxen

Figure S3. Confirmation of drug-induced luminescence phenotypes using RT-qPCR. CK1 inhibitor SSR112050 increased period length of per3 (A) and per1b (B) circadian oscillations. Anti- inflammatory naproxen increased amplitude of per3 (C) and per1b (D) circadian oscillations. Mean ± SEM is shown. *P<0.05 by ANOVA with Tukey’s test. A B Leukotriene receptor inhibitor DMSO 8 Pranlukast 1.2 4 1.0 0.8 * Amplitude Period Phase phase Δ Net 0 0.6 AICAR AMPK activator -4 0.4 Betulinic Acid NF-kB activator -8 50-22-6 glucocorticoid 0.2 Amplitude (RLU) Dexamethasone Na glucocorticoid -12

Luminescence (a.u.)Luminescence 0.0 Prednisolone glucocorticoid 24 48 72 96 DMSO 30 μM Adapalene retinoid Time (h) Pranlukast

Cyclosporin immunosuppressant Pravadoline NSAID SC 51322 EP1 receptor antagonist SU-6656 Src kinase inhibitor Ibuprofen NSAID Nimodipine L-type Ca channel inhibitor Tivozanib VEGF inhibitor Piroxicam NSAID Denbufylline PDE inhibitor SC 19220 EP1 receptor antagonist SU-6656 Src kinase inhibitor Chloroxine antibacterial Oxaprozin NSAID SU-6656 Src kinase inhibitor Meloxicam NSAID Rapamycin mTOR inhibitor WIN 62577 NK1 receptor antagonist Rapamycin mTOR inhibitor Ibuprofen NSAID 3 2 2 0 1 2 3

S.D. - - -

Figure S4. Drugs that affected Tg(per3:luc) luminescence circadian amplitude. (A) Clusters of drugs that affected circadian amplitude by at least 3 standard deviations from the mean of same- plate DMSO controls is shown. (B) Leukotriene receptor inhibitor pranlukast decreased circadian amplitude. *P<0.05 by ANOVA with Tukey’s test. A B PDE inhibitor DMSO glucocorticoid 15 2.0 Denbufylline NSAID 10 *** PDE inhibitor 1.5 5 other anti-inflammatory 0 1.0 antibiotic

Amplitude Period Phase phase Δ Net AMPK activator -5 0.5 -10 Melengestrol Amplitude (RLU)

Warfarin (a.u.)Luminescence -15 0.0 24 48 72 DMSO 30 μM Cortisone acetate Time (h) Prednisolone Denbufylline Dicumarol Oleic acid Dexamethasone C Pro-inflammatory: gut Metformin 10 DMSO 1.2 AICAR TNBS 1.0 Corticosterone 5 TMB HCl 0.8 *** SB415286 0 0.6 Adapalene 0.4 AICAR -5 0.2 SU-6656 Amplitude (RLU) Cyclosporin -10 0.0 Luminescence (a.u.)Luminescence 24 48 72 96 DMSO 50 μM Pravadoline TNBS SC 51322 Time (h) SU-6656 Ibuprofen DMSO Nimodipine D Retinoid Adapalene Tivozanib 15 2.0 ** Piroxicam 10 Denbufylline 1.5 SC 19220 5 SU-6656 0 1.0 Chloroxine -5 Oxaprozin 0.5 SU-6656 -10 Amplitude (RLU)

Meloxicam (a.u.)Luminescence -15 0.0 Rapamycin 24 48 72 96 DMSO 30 μM WIN 62577 Time (h) Adapalene Rapamycin Ibuprofen Polymyxin B sulfate E FAK inhibitor PF562271 DMSO 20 2.0 Dibucaine HCl PF562271 ** 15 Motesanib 1.5 Salmeterol xinafoate 10 Rapamycin 5 1.0 Naproxen 0 -5 Ibuprofen 0.5 -10

Adapalene Amplitude (RLU)

Estradiol acetate (a.u.)Luminescence -15 0.0 24 48 72 96 DMSO 10 μM Cyproterone Acetate Time (h) PF562271 Pravadoline Nalidixic acid sodium AV951 VEGFR inhibitor IV Tivozanib Chloroxine 3 2 2 0 1 2 3

S.D. - - -

Figure S5. Inflammatory drugs that affected Tg(per3:luc) luminescence circadian amplitude. (A) A cluster of drugs that affected circadian amplitude by at least 3 standard deviations from the mean of same-plate DMSO controls is shown. Color-coding indicates drug classes. Anti- inflammatory compounds that increased circadian amplitude include PDE inhibitor denbufylline (B), retinoid adapalene (D) and FAK inhibitor PF562271 (E). TNBS, which induces inflammation in the gut, decreased circadian amplitude (C). **P<0.01, ***P<0.001 by ANOVA with Tukey’s test. Amplitude Period Phase phase Δ Net

Tolterodine antimuscarinic Chlorpheniramine H1R antagonist Sorafenib EGFR inhibitor Canrenone aldosterone antagonist Chenodiol cholesterol synthesis inhibitor Tacrolimus FKBP ligand Oxaprozin NSAID Aminopyrine analgesic Flutamide anti-androgen Butaclamol dopamine receptor antagonist ALX 5407 inhibitor Manidipine calcium channel blocker Verapamil calcium channel blocker Paroxetine SSRI Butaclamol dopamine receptor antagonist Atropine antimuscarinic Citalopram SSRI Flufenamic Acid NSAID Clozapine dopamine receptor antagonist Duloxetine SNRI Taurine Rifaximin antibiotic CAS 879127-07-8 EGFR inhibitor Vincristine microtubule assembly inhibitor Vinblastine microtubule assembly inhibitor Amsacrine topoisomerase inhibitor Lorglumide CCK receptor antagonist Psora-4 Kv1.3 channel inhibitor Gefitinib EGFR inhibitor Benzydamine NSAID Chlorphenyl dichloroethane antiadrenal

Clomipramine tricyclic antidepressant Nisoldipine calcium channel blocker Tyrphostin 8 calcineurin inhibitor Vinpocetine PDE inhibitor Tranylcypromine MAOI Suloctidil platelet aggregation antagonist CGP 13501 GABA-B enhancer L703606 NK1 tachykinin antagonist Dofetilide potassium channel blocker GW-7647 PPAR alpha agonist Pimozide dopamine receptor antagonist Tolcapone COMT inhibitor Prasterone glucocorticoid Acetyl farnesyl cysteine fanesylation inhibitor Aprindine hERG channel blocker Efavirenz reverse transcriptase inhibitor CyPPA potassium channel activator Chlorophyllide antineoplastic JFD00244 SIRT inhibitor Ivermectin nicotinic ACh receptor modulator Clinafoxacin antibiotic Oxybutynin Hydroxycampothecin antineoplastic Chlorpheniramine H1R antagonist Chlorprothixene D2 receptor antagonist Lumiracoxib carboxylic acid NSAID Sertraline SSRI Roxithromycin antibiotic 3 2 2 0 1 2 3

S.D. - - -

Figure S6. Clusters of drugs that affected Tg(per3:luc) luminescence circadian phase. Drugs that affected circadian phase by at least 3 standard deviations from the mean of same-plate DMSO controls are shown. per3:luc B A Dunnett’s is severely amplitude cells, larvae, derived Figure Amplitude (RLU)

shown Period (h) 10000 12000 14000 16000 18000 2000 4000 6000 8000 20 30 40 50 60 10 0 0 while including S DMSO DMSO from . reduced 7 test ** 0.1 μM SSR112050 0.1 μM SSR112050 . *** in μ P Effects 1 M SSR112050 1 μM SSR112050 others, period increase ** period increase Tg(per < . 1 μM A002195858 1 μM A002195858 *** Tg(per 0 Labels 10 μM A002195858 10 μM A002195858 . *** 01

SSR 0.1 μM PF670462 0.1 μM PF670462 *** luminescence

, 1 μM PF670462 1 μM PF670462 3 of such *** 10 μM resveratrol : 10 μM resveratrol 3 luc) 112050 are

: 10 μM AICAR P 10 μM AICAR drugs luc)

< 1 μM LY294002 1 μM LY294002 0 colored as larvae 10 μM LY294002 10 μM LY294002 . 001

embryos 10 μM cyclosporin 10 μM cyclosporin , resveratrol, 10 μM piroxicam on 10 μM piroxicam A 10 μM indomethacin

for 10 μM indomethacin 002195858 amplitude

did 10 μM naproxen according luminescence 10 μM naproxen

pairwise 10 μM 10 μM sildenafil

not 10 μM

. 10 μM cilostamide

( 10 μM prednisolone 10 μM prednisolone A amplitude increase amplitude increase 10 μM SU6656 induce 10 μM SU6656 ) AICAR

Some 10 μM SC51322 was 10 μM SC51322 to and comparisons 10 μM PF04418948 10 μM PF04418948 10 μM PF562271 drug 10 μM PF562271

associated 10 μM Y11 10 μM Y11 similar PF circadian drugs and 10 μM betulinic acid 10 μM betulinic acid 670462 effects 10 μM imiquimod 10 μM imiquimod 1 μM L703606 1 μM L703606 LY 1 μM WIN62577 1 μM WIN62577 that phenotypes

294002 1 μM 1 μM toceranib between in , with 10 μM SU4312 10 μM SU4312 period increased induced 10 μM AM580

Tg(per 10 μM AM580 10 μM nalidixic acid 10 μM nalidixic acid

drug 10 μM μ , 10 M oxolinic acid

did 10 μM kasugamycin 10 μM kasugamycin each 3 length 10 μM BAYX1005

in 10 μM BAYX1005 - : amplitude amplitude decrease similar induced decrease luc) 10 μM LY255283 10 μM LY255283 not period per 10 μM cinalukast 10 μM cinalukast drug

. 1 μM Ro1069920 larvae 1 μM Ro1069920 3 and ( : 10 μM DSS 10 μM DSS luc B phenotypes 10 μM CuSO4 10 μM CuSO4 and ) toxicity length 1 μM 1 μM fluphenazine Drugs cells . amplitude 10 μM taurine 10 μM taurine phase advance phase advance DMSO 10 μM ALX5407 10 μM ALX5407

. 10 μM chlorpheniramine

. μ in 10 M chlorpheniramine Each Mean 10 μM pyrilamine

that 10 μM pyrilamine Tg(per 10 μM psora4 10 μM psora4 in control 1 μM CP339818 1 μM CP339818 per affected in 10 μM phloretin case 10 μM phloretin ± 10 μM LY367265

3 10 μM LY367265 cells SEM 3

: 1 μM

luc) 1 μM imipramine : luc by of per1b:luc B A Dunnett’s * reduced in while including per Figure P Amplitude (RLU) <

Tg(per Period (h) 6000 7000 8000 9000 1000 2000 3000 4000 5000 1 0 10 20 30 40 b . 0 0 05 others, : luc

, DMSO DMSO *** S period increase period increase circadian 3 ** 0.1 μM SSR112050 0.1 μM SSR112050 8 SSR *** test :

. 1 μM A002195858 1 μM A002195858 P zebrafish luc) *** 0.1 μM PF670462 0.1 μM PF670462 < Effects such .

0 10 μM resveratrol 10 μM resveratrol 112050 Labels . 10 μM AICAR 10 μM AICAR 01 larvae 1 μM LY294002 1 μM LY294002

, 10 μM LY294002 10 μM LY294002 as amplitude *** 1 μM cyclosporin 1 μM cyclosporin ***

of 10 μM cyclosporin 10 μM cyclosporin , are cells resveratrol, P

did 10 μM piroxicam 10 μM piroxicam A <

002195858 10 μM indomethacin 10 μM indomethacin 0 drugs colored μ μ

. 10 M naproxen 10 M naproxen not 001 . 10 μM sildenafil 10 μM sildenafil

( 10 μM cilostamide 10 μM cilostamide A was 10 μM prednisolone 10 μM prednisolone induce ) for 1 μM SU6656 1 μM SU6656 on Some

according 10 μM SU6656 10 μM SU6656 AICAR pairwise associated 1 μM SC51322 1 μM SC51322 amplitude increase amplitude increase luminescence and 10 μM SC51322 10 μM SC51322 10 μM PF04418948 10 μM PF04418948 similar

drugs 10 μM PF562271 10 μM PF562271

PF 10 μM Y11 10 μM Y11 and 1 μM betulinic acid 1 μM betulinic acid comparisons to 670462 10 μM betulinic acid 10 μM betulinic acid

phenotypes 1 μM imiquimod 1 μM imiquimod LY that drug with 10 μM imiquimod 10 μM imiquimod

294002 1 μM L703606 1 μM L703606 1 μM WIN62577 1 μM WIN62577 increased effects ,

drug 10 μM WIN62577 10 μM WIN62577 circadian induced

1 μM toceranib 1 μM toceranib *** 10 μM toceranib 10 μM toceranib , - 10 μM SU4312 10 μM SU4312 induced between did 1 μM AM580 1 μM AM580 ** in in 10 μM AM580 * 10 μM AM580 **

Tg(per 1 μM torin1 1 μM torin1 per not similar period 1 μM nalidixic acid 1 μM nalidixic acid

period 10 μM nalidixic acid 10 μM nalidixic acid . 1

: 10 μM oxolinic acid 10 μM oxolinic acid ( toxicity luc each B 10 μM kasugamycin 10 μM kasugamycin 3 ) 10 μM BAYX1005 10 μM BAYX1005 : amplitude amplitude decrease decrease phenotypes length luc) Drugs

cells 10 μM LY255283 10 μM LY255283 10 μM cinalukast 10 μM cinalukast length drug

. 1 μM Ro1069920 1 μM Ro1069920 larvae Mean . 10 μM DSS 10 μM DSS Each

that 10 μM CuSO4 10 μM CuSO4 in

and 1 μM fluphenazine 1 μM fluphenazine **

Tg(per 10 μM fluphenazine 10 μM fluphenazine . and

affected 10 μM taurine 10 μM taurine ± in phase advance phase advance DMSO

case 10 μM ALX5407 10 μM ALX5407 SEM

per 10 μM chlorpheniramine 10 μM chlorpheniramine

amplitude 10 μM pyrilamine 10 μM pyrilamine 3

: 1 μM psora4 1 μM psora4 1 luc) of

b 10 μM psora4 10 μM psora4 * control is amplitude : 1 μM CP339818 1 μM CP339818 luc severely 10 μM phloretin 10 μM phloretin shown larvae, 1 μM LY367265 1 μM LY367265

cells, 10 μM LY367265 10 μM LY367265 1 μM imipramine 1 μM imipramine by in . Bmal1:dluc

A 45 ** 40 ** 35 ** ** 30 25 20 15

Period (h) 10 5 0 M Y11 M DMSO M DSS M μ μ M torin 1 torin M M AICAR M M taurine M psora4 M M AM580 M M CuSO4 M μ μ SU6656 M SU4312 M μ μ μ μ phloretin M M sildenafil M L703606 M M toceranib M ALX5407 M M naproxen M SC51322 M μ μ M piroxicam M μ M cinalukast M μ μ M LY294002 M 2.65 imiquimod M LY255283 M pyrilamine M LY367265 M M PF670462 M PF562271 M μ μ M resveratrol M WIN62577 M CP339818 M μ μ M longdaysin M 7.94 7.94 μ BAYX1005 M BAYX1005 M M imipramine M M cyclosporin M cilostamide M μ μ μ μ μ μ μ μ Ro1069920 M μ μ μ M SSR112050 M μ M oxolinic acid oxolinic M μ μ μ μ μ 0.10 M A002195858 M acid nalidixic M fluphenazine M M PF04418948 M M prednisolone M μ M betulinic acid betulinic M 7.94 7.94 kasugamycin M 7.94 M indomethacin M 7.94 μ μ 2.65 2.65 μ μ μ μ μ μ 7.94 7.94 μ 71.43 μ 7.94 7.94 7.94 7.94 7.94 2.65 2.65 7.94 7.94 7.94 23.81 7.94 7.94 2.65 2.65 7.94 7.94 7.94 0.88 0.88 7.94 2.65 2.65 23.81 0.88 0.88 23.81 23.81 7.94 7.94 M chlorpheniramine M 2.65 7.94 7.94 7.94 23.81 23.81 7.94 7.94 23.81 23.81 0.10 0.10 23.81 23.81 7.94 7.94 μ 7.94 7.94 7.94 7.94 7.94 7.94 7.94 7.94 7.94 71.43 71.43 71.43 71.43 23.81 23.81 7.94 7.94 period increase amplitude increase amplitude phase advance decrease B 140

120

100

80 60 * 40 ** 20

Amplitude (RLU) 0 M Y11 M DMSO M DSS M μ μ M torin 1 torin M M AICAR M M taurine M psora4 M M AM580 M M CuSO4 M μ μ SU6656 M SU4312 M μ μ μ μ phloretin M M sildenafil M L703606 M M toceranib M ALX5407 M M naproxen M μ SC51322 M μ M piroxicam M μ M cinalukast M μ μ M LY294002 M 2.65 imiquimod M LY255283 M pyrilamine M LY367265 M M PF670462 M PF562271 M M resveratrol M WIN62577 M μ μ μ μ CP339818 M M longdaysin M 7.94 7.94 μ M BAYX1005 M BAYX1005 M imipramine M M cyclosporin M cilostamide M μ μ μ μ μ μ μ μ Ro1069920 M μ μ μ M SSR112050 M μ M oxolinic acid oxolinic M μ μ μ μ μ 0.10 M A002195858 M acid nalidixic M fluphenazine M M PF04418948 M M prednisolone M μ M betulinic acid betulinic M 7.94 7.94 kasugamycin M 7.94 7.94 M indomethacin M 7.94 μ μ 2.65 μ μ μ μ μ μ 7.94 7.94 μ 71.43 μ 7.94 7.94 7.94 7.94 7.94 2.65 2.65 7.94 7.94 7.94 23.81 7.94 7.94 2.65 2.65 7.94 7.94 7.94 0.88 0.88 7.94 2.65 2.65 0.88 23.81 23.81 0.88 23.81 23.81 7.94 7.94 M chlorpheniramine M 2.65 7.94 7.94 7.94 23.81 23.81 7.94 7.94 23.81 23.81 0.10 0.10 23.81 23.81 7.94 7.94 μ 7.94 7.94 7.94 7.94 7.94 7.94 7.94 7.94 7.94 71.43 71.43 71.43 71.43 23.81 23.81 7.94 7.94 period increase amplitude increase amplitude phase advance decrease Figure S9. Effects of drugs on luminescence circadian period length and amplitude in Bmal1:dluc human U2OS cells. (A) Some drugs that increased period length in Tg(per3:luc) larvae, including SSR112050, A002195858 and PF670462, induced similar phenotypes in Bmal1:dluc cells, while others, such as resveratrol, AICAR and LY294002, did not. Longdaysin, a CK1 and ERK2 antagonist (Ref 5), was used as a positive control. (B) Drugs that affected amplitude in Tg(per3:luc) larvae did not induce similar phenotypes in Bmal1:dluc cells. Each case of severely reduced circadian amplitude was associated with drug-induced toxicity. Mean ± SEM is shown. *P<0.05, **P<0.01 for pairwise comparisons between each drug and DMSO control by Dunnett’s test. Labels are colored according to drug effects in Tg(per3:luc) larvae.