High-throughput assays for sirtuin enzymes: A microfluidic mobility shift assay and a bioluminescence assay

Analytical Biochemistry 378 (2008) 53–59

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High-throughput assays for sirtuin enzymes: A microfluidic mobility shift assay and a bioluminescence assay Yichin Liu a,1,*, Raphaele Gerber b, John Wu a, Trace Tsuruda a, John D. McCarter a a b

Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA Caliper Life Sciences, 605 Fairchild Drive, Mountain View, CA 94043, USA

a r t i c l e

i n f o

Article history: Received 24 January 2008 Available online 4 March 2008 Keywords: SIRT HTS Mobility shift Bioluminescence NAD+ Deacetylase

a b s t r a c t Silent information regulator or sirtuin (SIRT) enzymes are b-nicotinamide adenine dinucleotide (oxidized) (NAD+)-dependent class III histone deacetylases. In this paper, two distinct assays to measure SIRT1 activity are described: a microfluidic mobility shift assay utilizing a fluorophore-labeled peptide substrate and a bioluminescence assay based upon quantitation of remaining NAD+. The mobility shift assay involves the electrophoretic separation of an N-acetyl-lysine-containing peptide substrate from deacetylated product which bears an additional positive charge. Interference from fluorescent compounds is minimized during screening by direct visualization of separated fluorophore-labeled substrate and product. A preferred peptide substrate for SIRT1 was identified using this assay. The NAD+ bioluminescence assay couples NAD+ consumption to the bacterial luciferase-catalyzed oxidation of decanal. This assay does not require synthesis of a labeled peptide and is applicable to sirtuins of any specificity with respect to peptide substrate. The stoichiometry between NAD+ consumption and peptide deacetylation was shown to be 1:1 by the NAD+ bioluminescence assay. Kinetic parameters of peptide and NAD+ cosubstrates and IC50 values of standard reference inhibitors determined in either assay were similar. With robust Z’ values (0.7), both assays are amenable to high-throughput screening. Ó 2008 Elsevier Inc. All rights reserved.

Silent information regulator (Sir)2 or sirtuins (SIRT) belong to the family of class III NAD+-dependant protein deacetylases [1,2]. To date, seven human isoforms have been identified (SIRT1 to SIRT7) [3] and found to be involved in alteration of chromatin structure, gene silencing, modulation of meiotic checkpoints, microtubule network, and metabolism (for reviews, see [3,4]). Consistent with the diverse cellular functions of sirtuins, different peptide substrates have been identified as targets for deacetylation by the various sirtuin enzymes. Activation of these enzymes has been thought to provide protection from certain neurodegenerative diseases and metabolic disorders, and the inhibition of the enzymes may inhibit progression of cancer [3,4]. Identification of small-molecule modulators (activators or inhibitors) of this class of enzymes could lead to the development of novel therapeutic agents. * Corresponding author. Fax: +(650) 467 6045. E-mail address: [email protected] (Y. Liu). 1 Current address: Genentech Inc., One DNA Way, South San Francisco, CA 94080, USA. 2 Abbreviations used: Sir, silent information regulator; SIRT, sirtuins; NAD+, bnicotinamide adenine dinucleotide (oxidized); NADH, b-nicotinamide adenine dinucleotide (reduced); FMN, riboflavin 50 -monophosphate; FMNH2, riboflavin 50 -monophosphate (reduced); LDH, L-lactate dehydrogenase; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; TFA, trifluoroacetic acid; HTS, high-throughput screening; RFU, relative fluorescence units; RLU, relative luminescence units; PoC, Percentage of control; BSA, bovine serium albumin. 0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.02.018

The NAD+-dependent deacetylation reaction catalyzed by sirtuins is as follows [5–10]: Sirtuin

protein-N-acetyl lysine þ NADþ ! N-acetyl-ADP-ribose þ protein-lysine þ nicotinamide: Recently, several assays to measure the enzymatic activity of sirtuins have been described. A popular assay method is the so-called Fluor de Lys fluorogenic assay (Biomol, Plymouth Meeting, PA) which utilizes a fluorophore conjugated to the carboxy terminal end of a short N-acetyl-lysine-containing peptide. Upon deacetylation, the unveiled free e-amino group of the lysine is a substrate for trypsin-catalyzed cleavage and release of the fluorophore, thus resulting in an increase in fluorescence [11,12]. Several potent SIRT1 inhibitors (including 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide: 1) have been identified from high-throughput screening using the Fluor de Lys assay [13]. However, recent data indicate that SIRT activation by some compounds in the Fluor de Lys assays does not correlate with that measured using either unlabeled peptide or full-length protein [14,15]. A fluorescence resonance energy transfer-based assay that utilizes N-acetyl-lysinecontaining peptides of various sequences flanked by a fluorophore and a quencher on opposite termini of the peptides has been reported by Marcotte et al. [16]. Several radioactive assays involving the use of either [3H]- or [14C]-labeled acetyl groups in the lysyl

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moiety of the peptide or in the nicotinamide moiety of NAD+ have also been developed to measure sirtuin activity [17]. In these assays, product and substrate were separated by either chromatography or filter binding and then detected by scintillation counting. Though sensitive, such radioactive assays pose drawbacks for large-scale screening including management of hazardous wastes. In this paper, two assays amenable to high-throughput screening (HTS) of sirtuins are described. The first assay is a fluorescencebased assay that utilizes Caliper’s mobility shift assay technology (Hopkinton, MA) [18]: specifically, the electrophoretic separation of N-acetyl lysine peptide substrate from the deacetylated product, which bears an additional positive charge. By allowing direct visualization of fluorophore-labeled separated substrate and product, this assay minimizes interference from fluorescent compounds during screening and does not require the use of coupling enzymes in detection [18,19]. The second assay is a bioluminescence assay that measures consumption of NAD+. The method combines the quantitative reduction of NAD+ to NADH [20,21] with the measurement of NADH using Photobacterium fischeri luciferase and NAD(P)H:FMN-oxidoreductase in the presence of long-chain aldehyde [22]. This assay does not require synthesis of a labeled peptide and is applicable to assay of sirtuins with any peptide substrate. Materials and methods Materials NAD+ was purchased from Calbiochem (San Diego, CA). FMN, glycine buffer solution containing 0.6 M glycine and 0.5 M hydrazine, and L-(+)-lactic acid and L-lactic dehydrogenase (LDH) of rabbit muscle type II were purchased from Sigma–Aldrich. NAD(P)H:FMN-oxidoreductase and luciferase from P. fischeri were purchased from Roche (Palo Alto, CA). Decanal, 98% was purchased from VWR International. Acetylated p53 peptide (Ac-p53) (HLKSKKGQSTSRH KK(Ac)LMFK) used in bioluminescence and HPLC assays was synthesized and purified by Quality Controlled Biochemicals (Hopkinton, MA). Fl-Ac-p53-peptide (fluoroscein-SKKGQSTSRHKK(Ac)LMFKTEGPDS), Fl-H4-K1Ac (fluoroscein-SGRGK(Ac)GGKGLGKGGAKRH RKVLR), Fl-H4-K3Ac (fluoroscein-SGRGKGGKGLGK(Ac)GGAKRHR KVLR), Fl-H4-K4Ac (fluoroscein-SGRGKGGKGLGKGGAK(Ac)RHRKV LR), and Fl-H4-K5Ac (fluoroscein-SGRGKGGKGLGKGGAKRHRK(Ac) VLR) used in mobility shift assays were synthesized and purified by Tufts University (Boston, MA). SIRT 1 inhibitor, 6-chloro2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide 1, was purchased from BioSPECS (The Netherlands) and Ambinter (France). Coating reagents 3 and 8 were purchased from Caliper Life Sciences (Hopkinton, MA). Sirtuin expression and purification Full-length His-tagged hSIRT1 was expressed in an Escherichia coli expression system. Following lysis of cells, clarified lysate was loaded onto a metal affinity column at 4 °C and eluted with an imidazole gradient. Pooled fractions were further purified by anion exchange liquid chromatography using a Q-Sepharose HP column. Protein was then concentrated prior to size-exclusion chromatography. Fractions containing hSIRT1 were pooled and the protein concentration was determined using the Bradford method using BSA as standard [23]. Purity of protein samples was determined to be >95% by SDS–PAGE under reducing conditions. The deacetylation activity of the hSIRT1 was confirmed using a published acetylated p53 peptide (Ac-p53) sequence [15] in HPLC assay. The hSIRT1 Km for Ac-p53 was determined to be 15.6 lM, consistent with the published data [15]. Proteins were dispensed into small aliquots and stored at 80 °C in buffer that contained

20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 10% glycerol, and 5 mM DTT. SIRT1 HPLC assay Various amounts of Ac-p53 peptide were incubated with 20 nM SIRT1 in reaction buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol) containing 500 lM NAD+. After 60-min incubation at room temperature, reaction was stopped by the addition of methanol to 28% final v/v. Samples were then analyzed by HPLC (System Gold; Beckman Coutler, Fullerton, CA) using a Phenomenex RP-C18 column (Phenomenex, Torrance, CA) run in a linear gradient of 0 to 32% acetonitrile in water containing 0.1% TFA over 20 min. The substrate and product were quantified by UV absorbance at 215 nm. NAD+-dependent deacetylation in microfluidic mobility shift assay SIRT1 reactions were carried out in a final volume of 50 lL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 25 lL 2X substrate to 25 lL of 2X enzyme, contained 4 nM SIRT1, 11.9 lM Fl-Ac-p53 peptide, 47 lM NAD+, 4 mM DTT, 100 mM Tris, pH 8.0, 0.003% Brij-35. In Km determinations, the appropriate substrate concentration was varied while the other cosubstrates were fixed at concentrations exceeding the respective Km values. After 90-min incubation at room temperature, the deacetylation reaction was stopped by the addition of 25 lL termination buffer (100 mM Tris, pH 8.0, 11% DMSO, 0.039% Brij-35, 0.3% coating reagent 3, 9 mM nicotinamide). The product and substrate in each independent reaction were separated using a 12-sipper microfluidic chip (Caliper Life Sciences) run on a Caliper LC3000 (Caliper Life Sciences). For Km determination, the reaction within a single well was sipped every 42 s for a total of 1 h and activity was measured in kinetic mode. The separation of product and substrate was optimized by choosing voltages and pressure using Caliper’s Optimizer software (Caliper Life Sciences). The separation buffer contained 100 mM Tris, pH 8.0, 5% DMSO, 0.015% Brij-35, 0.1% coating reagent 3, 1X coating reagent 8. For Fl-Ac-p53-peptide, the separation conditions used a downstream voltage of 2300 V, an upstream voltage of 500 V, and a screening pressure of 0.8 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electrophoregram using HTS Well Analyzer software (Caliper Life Sciences). NAD+-dependent deacetylation in bioluminescence assay SIRT1 reactions were performed in a final volume of 30 lL per well in a 384-well microplate. A standard SIRT1 reaction contained 10 nM enzyme, 25 lM NAD+, 25 lM Ac-p53 in SIRT1 reaction buffer (100 mM Tris–HCl, pH 8.0, 20 mM NaCl, 4 mM DTT,100 lg/mL BSA, 0.003% Brij-35), except in Km determinations when the appropriate substrate was varied while the other cosubstrate was fixed at concentrations exceeding the respective Km values. To ensure that time-dependent modulators were detected, 20 lL 1.5X SIRT1 was first incubated with 0.5 lL of 1 mM compounds or DMSO for 30 min at room temperature. The enzyme reaction was initiated, using a Multidrop (Thermo Electron Corp., Waltham, MA), by addition of 10 lL of a 3X stock solution containing NAD+ and Ac-p53 to the enzyme/compound mixture in a Greiner (Frickenhausen, Germany) black V-bottomed polypropylene 384-well plate. After incubation for 2 h at ambient temperature, the reaction was quenched by the addition of 10 lL of LDH reagent containing 5 mM nicotinamide, 10 mM lactic acid, 62.5 U/mL LDH, 600 lM glycine, 500 lM hydrazine. The conversion of NAD+ to NADH by LDH was carried out for 1 h at ambient temperature. NADH can be quanti-

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fied by its intrinsic fluorescence. The fluorescence emission was detected using Tecan Safire II (San Jose, CA) with excitation at 340 nm and emission at 440 nm. Using a Multimek automated pipettor (Beckman-Coulter, Fullerton, CA), 4 lL of the SIRT1/LDH mixture was added into 50 lL of luminescence reaction mixture (50 mM Hepes, pH 7.5, 1 mM DTT, 1.5 mM EDTA, 100 lg/mL BSA, 10 lg/mL luciferase, 0.05 U/mL NAD(P)H:FMN-oxidoreductase, 500 lM decanal, 5 lM FMN) in Costar white nontreated flat-bottomed polystyrene plates (Corning, Corning, NY). Due to the fast decay of luminescent intensity, luminescence in the reaction was measured within 15 min using LEADseeker Imaging Systems (GE Healthcare, Pittsburgh, PA) with 5 s of exposure time. The positive control for each plate was SIRT1 reaction in the presence of DMSO and the negative control was reaction mixture without enzyme. Enzyme kinetics and inhibition analysis Determination of the kinetic parameters, Km and Vmax, were performed by incubating enzyme (4 nM SIRT1 in microfluidic mobility shift assay or 20 nM SIRT1 in NADH fluorescence assay) for 1 h. Substrate conversion was plotted against substrate concentration and the Km value was determined by a nonlinear regression fit to the Michaelis–Menten equation using GraphPad Prism (GraphPad Software Inc., San Diego, CA). IC50 determinations of inhibitors were performed by incubating enzyme (4 nM SIRT1 in microfluidic mobility shift assay or 10 nM SIRT1 in NAD+ bioluminescence assay) with various inhibitor concentrations serially diluted two-fold in assay buffer. The plate was incubated at ambient temperature before addition of respective reaction termination buffers and analysis by four-parameter logistic nonlinear regression using GraphPad Prism. Percentage of control (PoC) values were calculated as PoC = 100  [(r  r of negative control)/(r of positive control  r of negative control)], where r is the observed signal. Percentage inhibition equals 100  PoC. Z’ factors were calculated using the following equation: Z’ = 1[3(standard deviation of negative control) + 3(standard deviation of positive control)]/[mean of positive control  mean of negative control] [24]. Results and discussion Microfluidic mobility shift assay Caliper’s microfluidic mobility shift assay technology utilizes pressure and voltage to separate substrates and products of enzymatic reactions. Quantitation of remaining substrate and newly generated product is achieved by measuring the fluorescence intensities of both product and substrate peaks (Fig. 1). During enzyme assays, substrate consumption is typically restricted to a maximum of 30–40%, a level empirically demonstrated to reflect initial rates and to not result in shifts in inhibitor IC50 values due to excessive substrate depletion. Separation of product and substrate depends on the differences in electrophoretic mobility contributed primarily by the charge of the molecule and secondarily by the mass of the species. This technology has been previously used to assay several kinase and phosphatase targets via separation of the appropriate substrates and their corresponding products [25– 27]. Herein this mobility shift assay is shown to effectively separate SIRT1 substrate peptides containing a neutral acetylated lysyl residue from product peptides containing a deacetylated lysine bearing an additional positive formal charge. Design of peptides for mobility shift assay Various proteins have been identified as targets of SIRT1 deacetylation activity in vitro. These proteins include p53, FOXO-1, histone H4, and PGC1-a. Several of these proteins have multiple

Fig. 1. SIRT1 reaction in microfluidic mobility shift assay. Representative electrophoregram demonstrating separation of deacetylated peptide product from substrate Fl-Ac-p53-peptide on Caliper LC3000.

acetylation sites [3,4]. To probe the preferred substrate specificity of human SIRT1, a panel of fluorescein-labeled 22mer peptides based on p53 and H4 sequences containing N-acetyl lysine residues were synthesized and purified to >95% purity (as judged by HPLC). Under standard conditions of 10 nM SIRT1, 500 lM NAD+, 100 lM peptide, deacetylation of 50% of this p53-derived peptide occurred within 40 min at ambient temperature. Under the same standard conditions, 25, 10, 10, and <5% deacetylation of 100 lM Fl-H4-K4Ac, Fl-H4-K5Ac, Fl-H4-K3Ac, and Fl-H4-K1Ac, respectively, were observed (Table 1). Thus, under these conditions, the p53-derived peptide containing the K382 acetyl lysine underwent SIRT1-catalyzed deacetylation most efficiently. SIRT1 activity characterized using mobility shift assay Using this p53-derived K382 N-acetyl lysine peptide, kinetic parameters of the fluorescein-labeled peptide and of NAD+ with SIRT1 were determined. Km values were found to be 11.9 ± 1.6 and 43 ± 8 lM, respectively (Fig. 2), values similar to those previously reported [15,16]. Using peptide and NAD+ concentrations similar to their respective Km values, the potencies of the SIRT1 inhibitors nicotinamide (Fig. 3A) and 6-chloro-2,3,4,9-tetrahydro1H-carbazole-1-carboxamide 1 (Fig. 3B) were also determined. Nicotinamide inhibited SIRT1 with an IC50 value of 88 ± 2 lM whereas compound 1 resulted in inhibition of the enzyme with an IC50 value of 165 ± 50 nM (Figs. 3C and 3D). Again, these IC50 values were in accord with reported values [13,16]. A repeat of the experiment using a 20-fold increase in NAD+ concentration ðto  20  K m NADþ Þ resulted in a 6-fold increase in IC50 for nicotinamide, whereas a 20-fold increase in peptide concentration ðto  13  K m peptide Þ did not result in any increase, within error,

Table 1 Relative deacetylation rates of different fluorescein-labeled acetylated peptides catalyzed by SIRT1 Peptide

Sequence

Relative Rate

FI-Ac-p53 FI-H4-K1Ac FI-H4-K3Ac FI-H4-K4Ac FI-H4-K5Ac

FI-SKKGQSTSRHKK(Ac)LMFKTEGPDS FI-SGRGK(Ac)GGKGLGKGGAKRHRKVLR FI-SGRGKGGKGLGK(Ac)GGAKRHKKVL R FI-SGRGKGGKGLGKGGAK(Ac)RHRKVLR FI-SGRGKGGKGLGKGGAKRHRK(Ac)VLR

1.00 <0.O5 0.10 0.25 0.10

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Fig. 2. Km determination for cosubstrates (A) NAD+ and (B) Fl-Ac-p53 peptide in microfluidic mobility shift assay. Km values for NAD+ and Fl-Ac-p53 were 43 ± 8 and 11.9 ± 1.6 lM, respectively. RFU, relative fluorescence units.

Fig. 3. Chemical structures of SIRT1 inhibitors (A) nicotinamide and (B) 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamid 1. Dose-response of (C) nicotinamide and (D) compound 1 in the microfluidic mobility shift assay. IC50 value was determined to be 88 ± 2 lM for nicotinamide whereas compound 1 yielded an average IC50 value of 165 ± 50 nM.

in the IC50 for nicotinamide. These results are consistent with nicotinamide acting as a noncompetitive inhibitor with respect to peptide and as a competitive inhibitor with respect to NAD+ [28] and are fully in accord with the available crystal structure showing nicotinamide bound at the NAD+ site [29]. NAD+ bioluminescence assay for SIRT1 Recent reports indicate that the aminomethylcoumarin moiety of the fluorogenic substrates used in the SIRT1 Fluor de Lys assay can affect the binding of other small molecules to the enzyme [14,15]. We therefore set out to develop an assay that utilizes label-free peptide substrate and is amenable to HTS. Current knowledge of the enzyme mechanism indicates that consumption of one molecule of NAD+ substrate corresponds to generation of one deacetylated peptide product [29,30]. NAD+ concentrations have been previously determined via reduction of nonfluorescent NAD+ to fluorescent NADH (excitation 340 nm, emission 440 nm) using various NAD+-dependent dehydrogenases [31]. However, the sensitivity of such assays is low (limited to NAD+ concentra-

tions in the double digit micromolar range) and the excitation and emission wavelengths used are prone to interferences from fluorescent compounds during screening. The conversion of NAD+ by LDH to NADH was coupled to a bioluminescence reaction to increase the assay sensitivity and obviate the need for detection using wavelengths associated with NADH fluorescence (Fig. 4A). In the first step, NAD+ was reduced to NADH by use of excess lactate to favor the forward direction of this reversible reaction, producing pyruvate and NADH. Hydrazine was added to irreversibly react with pyruvate to generate pyruvate hydrazone and to drive the quantitative reduction of NAD+ to NADH [20,21]. The NADH produced was then utilized in the NAD(P)H:FMN-oxidoreductase-catalyzed reaction in reducing FMN to FMNH2. FMNH2 was then oxidized to FMN by bacterial luciferase to produce decanoic acid from decanal [22]. Initially, all of the assay reagents and coupling enzymes were included in the assay following quenching of the SIRT1 reaction by addition of 5 mM nicotinamide. However, it was found that hydrazine inhibits the luciferase and NAD(P)H:FMN-oxidoreductase enzymes (data not shown). Thus, the minimum amount of hydrazine re-

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Fig. 4. (A) Reaction scheme of SIRT1 NAD bioluminescence assay. The sensitivity of NADH fluorescence assay is in lM range whereas the luminescence assay is able to detect nM NAD+ level. Representative plate views of bioluminescent signal (high NAD+ level in red and low NAD+ level in blue) for (B) single-point assay plate and (C) dose-response assay for SIRT1 inhibition. Column 23 (HI control) contained enzyme and substrates and column 24 (LO control) contained substrates but no enzyme. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

quired to result in quantitative and irreversible NAD+ reduction was empirically determined, followed by dilution of the SIRT1 and LDH reaction mixture into the NAD(P)H:FMN-oxidoreductase and luciferase mixture to initiate the luminescence reaction. The bioluminescent signal from P. fischeri luciferase has a half-life of 10 min (data not shown). Use of this dilution step to initiate the coupling enzyme reaction permits better management of the timing of reagent additions and detection. Still, the relatively fast decay of the bioluminescence signal necessitates the use of imaging-based detection systems to minimize signal drift within a plate which could otherwise confound results. Examples of a primary screening plate and a dose-response assay plate are shown in Figs. 4B and 4C, respectively. Under these conditions, a titration of NAD+ converted by the LDH/hydrazine reaction was shown to produce the same luminescence signal as a titration of NADH directly, suggesting that NAD+ was quantitatively converted to NADH by the coupling system (Fig. 5A). Based on the substrate preference analysis from the mobility shift assay, a label-free acetylated peptide based on the K382 p53 sequence (Ac-p53) [15] was synthesized and purified for use in the bioluminescence assay. A titration of Ac-p53 against 50 lM NAD+ using 40 nM SIRT1, measured upon completion of the reaction, confirmed that the stoichiometric ratio between the consumption of NAD+ and the deacetylation of peptide is 1:1 (Fig. 5B), a result fully consistent with the presumed catalytic mechanism [29,30]. SIRT1 activity characterized by NADH fluorescence and NAD+ bioluminescence assays The Km values for the cosubstrates NAD+ and Ac-p53 were determined using an NADH fluorescence assay. In the presence of saturating (500 lM) NAD+ the Km for Ac-p53 was found to be 17 ± 3 lM, and in the presence of saturating (300 lM) Ac-p53 pep-

Fig. 5. (A) Titration of coupling-system-derived NAD+ (d) and NADH (s) from standard curve in bioluminescence assay. Concentrations shown are the amounts of NAD+ and NADH present in the final bioluminescence detection mixture. (B) Titration of Ac-p53 peptide against 50 lM NAD+ upon completion of the sirtuin-catalyzed reaction (40 nM SIRT1). RLU, relative luminescence units.

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tide the Km for NAD+ was found to be 61 ± 12 lM, values consistent with those obtained in the mobility shift assay. IC50 values for two SIRT1 inhibitors were determined using the NAD+ bioluminescence assay: for nicotinamide IC50 = 81 ± 18 lM and for carboxamide inhibitor 1 IC50 = 125 ± 21 nM. These values are consistent with those determined by the microfluidic mobility shift assay (Figs. 6A and 6B). Thus the two methods result in determination of similar substrate kinetic parameters and inhibitor IC50 values with SIRT1. High-throughput screening of SIRT1 Because pharmaceutical library samples are typically dissolved in DMSO, the tolerance of SIRT1 activity to DMSO concentration

was determined. With 15 nM SIRT1, 25 lM NAD+, and 25 lM Acp53 peptide, no significant decrease in enzyme activity was observed up to a DMSO concentration of 6.7% in the NAD+ bioluminescence assay (Fig. 7A). The DMSO concentration used under high-throughput screening conditions was 1.7%. A screening pilot study of several plates was carried out using the bioluminescence assay. Assay wells included 10 nM SIRT1, 25 lM NAD+, 25 lM Ac-p53 peptide incubated for 2 h at ambient temperature in 384-well microplates. Plate positive controls in the reaction contained enzyme and substrates, whereas plate negative controls contained substrates but no enzyme. Consumption of NAD+ in positive controls was 35% (S/B 1.6), mean Z’ for all plates was 0.69, and throughput was estimated at >50,000 wells/ day. The distribution of positive and negative controls representing

Fig. 6. Potencies of nicotinamide (A) and compound 1 (B) were determined in dose-response using the NAD+ bioluminescence assay (IC50 = 81 ± 18 lM for nicotinamide and IC50 = 125 ± 21 nM for carboxamide inhibitor 1).

Fig. 7. (A) Effect of DMSO concentration on SIRT1 activity determined using NAD+ bioluminescence assay. DMSO was titrated in a reaction containing 15 nM SIRT1, 25 lM NAD+, 25 lM Ac-p53 peptide. Distribution of control wells in (B) NAD+ bioluminescence assay and (C) microfluidic mobility shift assay. Control wells in the presence and the absence of SIRT1 enzyme. Positive controls (HI, top circles) consisted of wells containing SIRT1, substrates, and DMSO in reaction buffer. Negative controls (LO, bottom circles) consisted of wells containing substrates and DMSO in reaction buffer. Mean Z’ for these test plates was 0.69 for NAD+ bioluminescence assay and 0.80 for the mobility shift assay.

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SIRT1 reaction and no enzyme control, respectively, in 10 representative plates for the bioluminescence assay is shown in Fig. 7B. Because the bioluminescence assay is proportional to the amount of NAD+ substrate remaining in the SIRT-catalyzed reaction, the signal arising from the positive control is expected to be less than that arising from the negative control, where no NAD+ has been consumed. Activator false positives could arise due to inhibition of one or more of the coupling enzymes or quenching of the luminescent signal by specific compounds, whereas activator false negatives could arise due to luminescent compounds. Conversely, inhibitor false positives could arise due to luminescent compounds, whereas inhibitor false negatives could arise due to inhibition of one or more of the coupling enzymes or quenching of the luminescent signal. Inhibition of the coupling enzymes is the most common of these scenarios, and activator false positives could be retested against the coupling enzymes in a SIRT-independent fashion. Similarly, a screening pilot study of several plates was carried out using the mobility shift assay. Assay wells included 4 nM SIRT1, 11.9 lM Fl-Ac-p53-peptide, 47 lM NAD+ incubated for 90 min in 384-well microplates at ambient temperature before the reactions were analyzed using a 12-sipper microfluidic chip. Plate positive controls in the reaction contained enzyme and substrates, whereas plate negative controls contained substrates but no enzyme. Under typical screening conditions, a maximum of 30% of the substrate was deacetylated (S/B 7). Mean Z’ value was 0.8 (Fig. 7C), and throughput was estimated at 10,000 wells/day. Conclusions This paper describes two distinct assays for NAD+-dependent deacetylases which were used to characterize SIRT1 activity and assess the potency of sirtuin inhibitors: a microfluidic mobility shift assay and a NAD+ bioluminescence assay. The mobility shift assay affords high-quality data with Z’ > 0.8 and minimal compound fluorescent interference but relatively lower throughput. Utilizing this assay, a preferred peptide substrate was identified and the kinetic parameters were determined. Km and IC50 values of two known small-molecule inhibitors were consistent with previously reported values. In the NAD+ bioluminescence assay, the stoichiometry of NAD+ consumption and peptide deacetylation was shown to be 1:1, and kinetic parameters and inhibitor potency were consistent with those determined using the mobility shift assay. Z’ values were 0.7, and throughput during HTS was >50,000 wells per day. Although either assay is amenable to HTS, the higher throughput of the bioluminescence assay favors its application in screening whereas the direct observation of deacetylated peptide and reduced potential compound interference in the mobility shift assay are an advantage in hit confirmation and mechanistic studies. Acknowledgments We thank Javier Farinas and Minghan Wang for stimulating discussions. References [1] R.A. Frye, Characterization of five human cDNAs with homology to the yeast SIR2 gene: Sir2-like proteins (sirtuins) metabolize NAD and may have protein ADP-ribosyltransferase activity, Biochem. Biophys. Res. Commun. 260 (1999) 273–279. [2] R.A. Frye, Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins, Biochem. Biophys. Res. Commun. 273 (2000) 793–798.

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