SIRT1 (IC50 >50 μM); SIRT2 (IC50 >50 μM); SIRT3 (IC50 >50 μM)

The sirtuins SIRT1, SIRT2, and SIRT3 are NAD(+) dependent deacetylases that are considered potential targets for metabolic, inflammatory, oncologic, and neurodegenerative disorders. Encoded library technology (ELT) was used to affinity screen a 1.2 million heterocycle enriched library of DNA encoded small molecules, which identified pan-inhibitors of SIRT1/2/3 with nanomolar potency (e.g., 11c: IC50 = 3.6, 2.7, and 4.0 nM for SIRT1, SIRT2, and SIRT3, respectively). Subsequent SAR studies to improve physiochemical properties identified the potent drug like analogues 28 and 31. Crystallographic studies of 11c, 28, and 31 bound in the SIRT3 active site revealed that the common carboxamide binds in the nicotinamide C-pocket and the aliphatic portions of the inhibitors extend through the substrate channel, explaining the observable SAR. These pan SIRT1/2/3 inhibitors, representing a novel chemotype, are significantly more potent than currently available inhibitors, which makes them valuable tools for sirtuin research [1].

ELT Affinity Selection [1]
Selections were carried out by capturing 2 μg of Flag-hSIRT3(118–399)-SBP on streptavidin matrix tips in the presence of (1) no β-NAD/no peptide substrate, (2) 100 μM β-NAD (Sigma), or (3) 20 μM TRSGKthioacetylVMRRLLR for three rounds. A no target control selection with buffer was carried out concurrently in the absence of SIRT3 protein. Streptavidin tips were prewashed in selection buffer: 50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% Tween-20, 0.1 mg/mL sheared salmon sperm DNA (sssDNA, Ambion), 0.1 mg/mL BSA, and 5 mM β-mercaptoethanol. In the first round of selection, 2 μg of Flag-hSIRT3(118–399)-SBP protein was immobilized on prewashed tips in the presence of (1) no β-NAD/no peptide substrate, (2) 100 μM β-NAD, or (3) 20 μM thioacetylated peptide. The tips were washed two times with buffer containing the corresponding cofactor and substrate when necessary. Pooled ELT libraries (5 nmol) were passed over the immobilized SIRT3 in the presence of the corresponding cofactor and substrate for 1 h at room temperature. The tips were washed eight times with selection buffer containing the corresponding cofactor and substrate and two times with BSA free selection buffer containing the corresponding cofactor and substrate. Bound molecules were heat eluted by passing BSA free selection buffer containing no cofactors and substrates over the tip at 72 °C for 10 min. The cooled heat elution was postcleared twice by passing the elution over streptavidin tips for 15 min to remove any denatured SIRT3 and matrix binders. Fresh BSA and sssDNA were added to all samples and corresponding cofactor and substrate was added to the elutions as needed. Round 2 was performed as described for round 1 using freshly immobilized SIRT3 on streptavidin tips in the presence of corresponding cofactor and substrate and postcleared round 1 output. Round 3 was performed as described for round 1 using freshly immobilized SIRT3 on streptavidin tips in the presence of corresponding cofactor and substrate and postcleared round 2 output with the exceptions that the last two washes and elution were with BSA-free and sssDNA-free selection buffer and the round 3 output was not postcleared. Quantitative PCR was used to quantitate the outputs from each round of selection. The round 3 output was sequenced using an Illumina sequencing platform.

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Protein Cloning, Expression, and Purification [1]
Human SIRT3-(118–399) was cloned into a proprietary modified pET21b vector. The protein was expressed in Escherichia coli BL21-Gold(DE3) cells as an N-terminal fusion to a hexahistidine affinity tag with an integrated TEV protease site. A single colony was inoculated in LB media containing 100 μg/mL ampicillin at 37 °C and swirled at 250 rpm until the A600 reached 0.3. The culture was then cooled to 18 °C and swirled at 250 rpm until the A600 reached 0.6–0.8. 1-(2-Isopropylthio)-β-d-galactopyranoside (IPGT) was added to a final concentration of 0.3 mM, and expression was continued at 18 °C and swirled at 160 rpm overnight. Cells were collected by centrifugation, and the pellet was resuspended in lysis buffer (200 mM NaCl, 5% glycerol, 5 mM 2-mercaptoethanol, and 25 mM HEPES-NaOH, pH 7.5) and sonicated to break the cells. The supernatant was separated from the cell debris by centrifugation at 10000g for 40 min at 4 °C and loaded onto a Ni-NTA column that was equilibrated with a buffer containing 200 mM NaCl, 5% glycerol, 5 mM 2-mercaptoethanol, 20 mM imidazole, and 25 mM HEPES-NaOH, pH 7.5. The column was washed with 5 column volumes of a buffer containing 200 mM NaCl, 5% glycerol, 5 mM 2-mercaptoethanol, 50 mM imidazole, and 25 mM HEPES-NaOH, pH 7.5, then eluted with a buffer containing 200 mM NaCl, 5% glycerol, 5 mM 2-mercaptoethanol, 250 mM imidazole, and 25 mM HEPES-NaOH, pH 7.5. The eluted protein was dialyzed in lysis buffer and digested with TEV protease at 4 °C overnight to remove the N-terminal His tag. The protein was loaded on a second Ni-NTA column equilibrated with lysis buffer. The untagged protein was eluted with a buffer containing 200 mM NaCl, 5% glycerol, 5 mM 2-mercaptoethanol, 5 mM imidazole, and 25 mM HEPES-NaOH, pH 7.5. The purified protein was dialyzed against a buffer containing 200 mM NaCl, 5 mM 2-mercaptoethanol, and 20 mM Tris-HCl, pH 8.0, and concentrated. The protein was further purified by elution with dialyzing buffer over a S200 column to 95% purity as assessed by SDS-PAGE analysis stained by Coomassie Brilliant Blue R-250 and concentrated to 10–15 mg/mL in the dialyzing buffer.

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SIRT1, SIRT2, and SIRT3 Biochemical Assays [1]
Deacetylation of a Trp 5-mer peptide (Ac-RHKKAcW-NH2) by His-SIRT1(1–747), His-SIRT2(1–389), and His-SIRT3(102–399) was measured by a discontinuous OAADPr mass spectrometry assay which measures OAADPr (2′-O-acetyl-ADP-ribose) production. All assays were performed at room temperature in reaction buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM DTT, 0.05% BSA). Test compounds (1 μL in DMSO) were preincubated with either SIRT1 (5 nM), SIRT2 (10 nM), or SIRT3 (5 nM) in reaction buffer (50 μL) for 20 min. For IC50 determination, Trp 5-mer peptide was added at KM conditions (2 μM for SIRT1, 10 μM for SIRT2, or 2.2 μM for SIRT3) along with NAD at KM (80 μM for SIRT1, 50 μM for SIRT2, and 130 μM SIRT3) for a final volume of 100 μL. The reaction was quenched after 30 min with 10 μL of stop buffer (50 mM nicotinamide in 10% formic acid) to give a final concentration of 0.9% formic acid and 4.5 mM nicotinamide. To prepare the assays for analysis, 20 μL of reaction volume was mixed in 80 μL of 50:50 acetonitrile/methanol mixture. The plates were analyzed on an Agilent RapidFire 200 high-throughput mass spectrometry system (Agilent, Wakefield) coupled to an AB Sciex API 4000 mass spectrometer fitted with an electrospray ionization source in negative MRM mode monitoring the transition 600.1/345.9 for the parent/daughter ion under low resolution conditions. Peak data was integrated using RapidFire Integrator software.

[1]. Discovery of thieno3,2-dpyrimidine-6-carboxamides as potent inhibitors of SIRT1, SIRT2, and SIRT3. J Med Chem. 2013 May 9;56(9):3666-79.

Using coding library techniques, molecules interacting with SIRT3 were enriched, and a novel class of highly effective SIRT1/2/3 pan-inhibitors was identified. Analysis based on ELT sequencing data revealed that the selected ring 3 structural unit (thieno[3,2-d]pyrimidine-6-carboxamide) is the key core framework for the inhibitory function of this chemotype. Preliminary non-DNA library construction confirmed that compounds 11a-d are highly effective SIRT1/2/3 pan-inhibitors. To improve the physicochemical properties of compound 11c, its molecular weight was reduced, ultimately leading to the selection of acetamide 20, which achieved a good balance between inhibitory activity and molecular weight reduction. Further structure-activity relationship studies showed that thioacetyl compounds 25, tert-butylamide 28, and sulfonamide 31 are particularly effective pan-inhibitors. To better understand the binding interactions, we crystallized complexes of the three pan-inhibitors (11c, 28, and 31) with SIRT3. Thieno[3,2-d]pyrimidine-6-carboxamide inhibitors bind in the active site cleft, between a large Rossmann fold and a small zinc-binding domain occupying the nicotinamide C pocket and substrate channel. Comparison with previously reported SIRT3 structures reveals a similar protein folding pattern, except for the flexible loop region where Phe157 forms a π-π stacking interaction with the thieno[3,2-d]pyrimidine core. The formamide group on the inhibitor is crucial for activity, forming key hydrogen-bonded interactions with residues in the nicotinamide binding pocket, similar to carba-NAD+ and the recently reported SIRT1/NAD+/43 ternary complex. The pan-inhibitor 11c and its truncated analogs 28 and 31 represent a significant advance in currently available sirtuin inhibitors. X-ray crystallography data confirm their binding patterns, which are in excellent agreement with the observed structure-activity relationship (SAR). The high efficiency of these inhibitors makes them valuable tools for understanding the biological effects resulting from the regulation of SIRT1, SIRT2, and SIRT3 deacetylase activity. [1]

1431411-18-5

Typically exists as solids at room temperature

1.6±0.1 g/cm3

479.6±40.0 °C at 760 mmHg

243.9±27.3 °C

0.0±1.2 mmHg at 25°C

1.74

1.06

4-chlorothieno[3,2-d]pyrimidine-6-carboxamide; 1431411-18-5; MFCD28098923; 4-chloro-Thieno[3,2-d]pyrimidine-6-carboxamide; CHEMBL2332049; SIRT-IN-6; SCHEMBL16011549; UGHXFHLQVJRWID-UHFFFAOYSA-N;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)