Supplementary Material for Pacholec, M. et al. manuscript SRT1720, SRT2183, SRT1460, and

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1 Supplementary Material for Pacholec, M. et al. manuscript SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1 Supplementary Figure Legends Figure S1. Determination of SIRT1 K m values for native p53 peptide and NAD +. The SIRT1 reactions were carried out at 5 nm SIRT1 under the conditions described under Experimental Procedures. For the K m value determinations of the native peptide, the peptide concentrations were varied from 0.78 to 100 M and NAD + was kept at 2 mm. For the K m value for NAD +, NAD + was varied from 0.5 to 1000 M and the native peptide was kept at 75 M. The data were fitted into the Michaelis Menten equation using KaleidaGraph. The K m and k cat values of 4.5 ± 0.17 M and 0.19 s -1 were obtained for the native peptide, respectively. The corresponding K m and k cat values for NAD + were 94 ± 5 M and 0.22 s -1. Figure S2. SDS-PAGE of purified p53. Purification of p53 using a Ni-NTA column followed by IEX Q Sepharose. Pools from each column were subjected to a 4-12% NuPage Bis-Tris SDS- PAGE under reducing conditions and stained with Coomassie Blue. lane 1-See Blue Plus 2 Markers; lane 2- pool from Ni-NTA (5 g); lane 3 pool from IEX Q Sepharose (1 g). Figure S3: SPR study on interaction of SRT1720 with p53-derived peptide substrates in the absence of SIRT1. Replicate injections of SRT1720 (2-fold dilutions at 3-50 M) passed over the surface of the TAMRA Peptide 1 (A) and the Native Peptide 1 (B). The amino acid sequence 1

2 of the TAMRA Peptide 1 and the Native Peptide 1 are identical and differ only in the TAMRA group (Table S1). 2

3 Figure S1 A. B Rate (nm/min) Rate (nm/min) [Peptide] ( M) [NAD] ( M) 3

4 Figure S2 MW kda

5 Figure S3 A 50 B Response, Ru Response, Ru Time (sec) Time (sec) 5

6 Table S1. The sequence of the peptides used in the present study. The TAMRA Peptides 1-2 are p53-derived peptides with the TAMRA group attached. The Native Peptides 1-3 are p53-derived peptides without the TAMRA group attached. The amino acid sequence of the Native Peptides 1-2 is identical to that in TAMRA Peptides 1-2. Peptides TAMRA-Peptide 1 TAMRA-Peptide 2 Native Peptide 1 Native Peptide 2 Native Peptide 3 Sequence (Biotin PEG3)-Ser-Thr-Ser-Ser-His-Ser-(Ac-Lys) Nle-Ser- Thr-Glu-Gly-Cys(TAMRA)-Glu-Glu-NH 2 Ac-Glu-Glu-Lys-Gly-Gln-Ser-Thr-Ser-Ser-His-Ser-(Ac-Lys)- Nle-Ser-Thr-Glu-Gly-Lys(TAMRA)-Glu-Glu-NH 2 (Biotin PEG3)-Ser-Thr-Ser-Ser-His-Ser-(Ac-Lys) Nle-Ser- Thr-Glu-Gly-Cys-Glu-Glu-NH 2 Ac-Glu-Glu-Lys-Gly-Gln-Ser-Thr-Ser-Ser-His-Ser-(Ac-Lys)- Nle-Ser-Thr-Glu-Gly-Lys-Glu-Glu-NH 2 Ac-Lys-Lys-Gly-Gln-Ser-Thr-Ser-Arg-His-Lys-(Ac-Lys)- Leu-Met-Phe-Lys-Thr-Glu-Gly-NH 2 6

7 Table S2. Receptors, enzymes/kinases, ion channels, and transporters assessed for the Sirtris compounds and resveratrol at 10 M. Receptors Enzymes/Kinases Adenosine (A 1, A 2a, A 3 ) Acetylcholinesterase Adrenergic (α 1, α 2A, α 2B, α 2C, β 1, β 2, β 3 ) ATPase Angiotensin-II (AT 1 ) ACE Cannabinoid (CB 1, CB 2 ) Carbonic Anhydrase II Chemokines (CXCR4) Caspase-3 Cholecystokinin (CCK A, CCK B ) COX1 Cytokines (TNFα) COX2 Dopamine (D1, D2S, D3, D4.4) MMP-9 Endothelin (ET A, ET B ) Monoamine Oxidase A GABA (GABA A, GABA B ) PDE11 Glutamate (AMPA, kainate, NMDA, glycine, PCP) PDE2 Histamine (H 1, H 2, H 3, H 4 ) PDE3 5-Hydroxytryptamine (5-HT 1A, 5-HT 1B, 5-HT 1D, PDE4 5HT 2A, 5HT 2B, 5-HT 2C, 5-HT 3, 5-HT 4e, 5-HT 6, 5-HT 7 ) Imidazoline PDE4 (rolipram binding) Leukotrienes (LTB4, LTD4) PDE5 Melanin Concentrating Hormone (MCH 1 ) PDE6 Melanocortin (MC 1, MC 3, MC 4 ) Abl Kinase Melatonin (MT1, MT3) CamK2α Kinase Motilin FLT-1 Kinase (VEGFR1) Muscarinic (M 1, M 2, M 3, M 4, M 5 ) Lyn Kinase Nicotinic (neuronal, muscle) p38α Kinase Neurokinin (NK1, NK2) ZAP70 Kinase Neuropeptide Y (Y1) Non-steroid nuclear receptors (PPARγ, Thyroid Hormone) Opioid (delta, kappa, mu) Sigma (non-selective) Somatostatin (sst4) Steroid Nuclear Receptors (Glucocorticoid, Androgen) Urotensin receptor (GPR14) Vasoactive intestinal peptide (VIP1) Vasopressin (V 1A, V 2 ) Ion channels Transporters Calcium channels; 5-HT L-type DHP Choline L-type (diltiazem) Dopamine L-type (verapamil) GABA N-type Norepinephrine Chlorine Channel Potassium Channel Calcium-dependent Sodium Channel (site 2) 7

8 Table S3. Selectivity profiles of SRT1720, SRT2183, SRT1460, and resveratrol. All four compounds were tested at 10 M against over 100 targets listed in Table S2. % inhibition is shown in colors designated below. ND, Not Determined. >80% Inhibition 51-80% Inhibition 50% Inhibition Target Class Target SRT1720 SRT2183 SRT1460 Resveratrol Receptors 5-HT1A HT1B HT1D HT2A HT2A (agonist site) HT2B (agonist site) HT2C HT2C (agonist site) HT HT4e HT HT A A2A A alpha alpha 2A alpha 2B alpha 2C AMPA Androgen AT beta beta beta CB CB CCKA (CCK1) CCKB (CCK2) CXCR D D2S D2S (agonist site) D D delta 2 (DOP) ETA ETB GABA-A GABA-A (BZD Site) GABA-B (1b) Glucocorticoid Glycine H H H H Imidazoline Kainate kappa (KOP) LTB4 (BLT1) LTD4 (CysLT1) M M

9 Target Class Target SRT1720 SRT2183 SRT1460 Resveratrol Receptors M M M MC MC MC MCH Motilin MT MT3 (ML2) mu (MOP) (agonist site) Nicotinic (muscle-type) Nicotinic (neuronal) NK NK NMDA PCP PPARgamma sigma sst TH TNF-alpha UT1 (GPR14) V1a V VIP1 (VPAC1) Y Enzymes ACE Acetylcholinesterase ATPase (Na+/K+) Carbonic anhydrase II 58 ND -7 8 Caspase COX COX MAO-A MMP ND PDE PDE PDE PDE PDE PDE Kinases Abl kinase CaMK2alpha FLT-1 kinase (VEGFR1) Lyn kinase p38alpha kinase ZAP70 kinase Ion Channels Ca2+ channel (L-type DHP) Ca2+ channel (L-type diltiazem) Ca2+ channel (L-type verapamil) Ca2+ channel (N-type) Cl- Channel ND 28 ND 22 K+ Channel (Ca2+ Dependent) Na+ channel (site 2) Transporters 5-HT transporter Choline transporter (CHT1) 94 ND 54 1 DA transporter GABA transporter NE transporter

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