GSK-3β(IC50 = 0.58 nM); GSK-3α(IC50 = 0.65 nM); cdc2(IC50 = 3700 nM)

Selisistat (1-10 μM) decreases the deacetylation activity of both human SirT1 and Drosophila Sir2 in transfected cells[1].

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Selisistat specificity in mammalian cells[1]
To determine the specificity and activity of selisistat on sirtuins, HEK293 cells were transfected with GCN5 (a histone acetyltransferase), and the nuclear factor kappa B (NFκB) p65 subunit (a characterized SirT1 substrate). GCN5 actively acetylates p65 as indicated by the ratio of acetylated p65 to total p65 protein in transfected cells. When human SirT1 is also co-transfected along with GCN5 and p65, the level of p65 acetylation is reduced by ∼80%. When Drosophila Sir2 is co-transfected into cells, the GCN5 acetylation of p65 is reduced by ∼70%. The addition of selisistat to these cells suppresses the SirT1 deacetylation restoring ∼50% of the p65 acetylation at 10 μm. Similarly, selisistat blocks the ability of Drosophila Sir2 to deacetylate p65 as indicated by the 60% recovery of the inhibited acetylation activity. These data show that selisistat inhibits the deacetylation activity of both Drosophila Sir2 as well as human SirT1.

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Selisistat is protective in cultured mammalian cell models of HD[1]
Given the robust positive effects of genetically reducing Sir2 on HD pathology in Drosophila, we sought to determine whether selisistat exhibited positive effects in mammalian models of HD. Rat pheochromocytoma cells (PC-12) expressing mHtt exon 1 fragments have been widely employed to study mHtt toxicity and aggregation. PC-12 cells inducibly expressing an exon 1 fragment of human Htt with an expanded polyglutamine repeat present with aggregates, transcriptional changes and cytotoxicity upon transgene expression. In this model, induction of mHtt expression results in a robust increase in toxicity (measured as lactate dehydrogenase [LDH] release), which was significantly reduced by treatment with selisistat at the concentrations of 1 and 10 μm.

In the R6/2 mouse model of Huntington's disease (HD), selenisistat (5 and 20 mg/kg, PO, daily; transgenic R6/2 mice commencing at 4.5 weeks of age until death) is protective[1].

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In this study, Selisistat (SEN0014196; EX 527) (5 µg/kg), administered to HFD rats twice a week for ten weeks, reduced the serum levels of triglyceride (TG), total cholesterol, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) and attenuated hepatic fibrosis evidenced by Masson's trichrome and hepatic fat by oil red-O staining. EX-527 upregulated SIRT2, SIRT3, and SIRT4 expression in the liver of HFD fed rats but downregulated transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) expression. It decreased proinflammatory cytokine production and hydroxyproline levels in the serum and SMAD4 expression and restored apoptotic protein (Bcl-2, Bax, and cleaved caspase-3) expression. These data propose a critical role for the SIRT4/SMAD4 axis in hepatic fibrogenesis. SIRT4 upregulation has the potential to counter HFD-induced lipid accumulation, inflammation, and fibrogenesis. We demonstrate that EX-527 is a promising candidate in inhibiting the progression of HFD-induced liver fibrosis.[3]

Class I and II HDAC Fluorimetric Assay. [2]
Class I and II HDAC deacetylase activities were measured in the above fluorimetric assay using a class I and II HDAC-containing HeLa cell extract and H4-K16(Ac) substrate representing residues 12−16 of histone H4 acetylated on lysine 16.

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Nicotinamide Release Assay. [2]
The activity of SIRT1 was measured in a nonfluorimetric assay using a p53 peptide substrate representing residues 368−386 acetylated on lysine 382. This assay measures the release of [14C]nicotinamide from [carbonyl-14C]-NAD, as previously described. Nicotinamide exchange was measured using the assay as described above in the presence of unlabeled nicotinamide added to a concentration of 52 μM. The added nicotinamide promotes release of [14C]nicotinamide from the labeled NAD through enzyme-catalyzed exchange. After release of [14C]nicotinamide from NAD, unlabeled nicotinamide binds to the enzyme and is converted to unlabeled NAD.
NAD glycohydrolase (NADase) enzymatic activity was measured in the nicotinamide release assay as described above. Crude NADase fraction from pig brain was purified by anion exchange chromatography. Each assay well contained 0.5 μg of purified enzyme and NAD at a concentration of 18.55 μM (70% of KM).

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Microsomal Stability. [2]
In vitro metabolic stability was assessed using rat hepatic microsomes. Compounds at a concentration of 10 μM were incubated at 37 °C with rat hepatic microsomes (1 mg of protein/mL) and quantified by HPLC/MS after 0, 5, 15, 30, and 60 min. Control incubations contained no microsomes.

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Cytochrome P450 Inhibition Assays. [2]
Cytochrome P450 assays were performed in a 384-well microplate format using recombinant human isozymes 3A4, 2D6, 1A2, 2C9, and 2C19 incubated with fluorogenic substrates as previously reported.

hERG Assay.[2]
Chinese hamster ovary (CHO) cells were stably transfected with the hERG potassium channel. Blockade of the hERG channel gives rise to a change in membrane potential that is measured using a potentiometric dye. Dye-loaded cells were incubated with 10 μM compound and 2 mM potassium chloride. Changes in fluorescence were measured in 384-well microplate format using a Tecan Safire fluorescence reader. The effect of compound on control CHO cells lacking the hERG channel was measured and used to correct for nonspecific quenching and toxicity.

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PC-12 exon 1-expressing mutant/wild-type Htt cell lines[1]
PC-12 7210 (exon 1 mutQ74) cells (PC12-Q74) stably expressing a GFP-tagged-exon 1 fragment of the human HD gene were obtained from Prof. Rubinsztein's laboratory (24). The tetracycline (Tet-on)-inducible mHTT construct comprises nucleotides 1–297 of the human Htt sequence (NM_00211) and includes a 74 CAG repeat expansion that, once expressed, is toxic to the cells. Cells were seeded in a 96-well poly-d-lysine (MW 70–150 kDa precoated plate at a density of 45K cells/100 μl medium/well in DMEM-containing 2% HS, 1% FBS, 100 mU/ml penicillin/streptomycin and 1% glutamax, then grown for 24 h prior to the experiment in an incubator at 37°C, with 90% of relative humidity and 10% CO2 atmosphere. The day of the experiment, the same medium but devoid of serum was added to the wells in order to obtain a final dilution of the previous serum concentration to 1:3. For transgene induction, the serum-free medium was complemented with doxycycline (final concentration 1 μg/ml). selisistat was added (from a DMSO 10 mm stock solution) to obtain the final concentrations described in the results, omitting its addition in the controls that received DMSO only. The final concentration of DMSO in all treatments and controls was 0.1%. At the 72 h time point, cell death was assessed by measuring levels of LDH released from cells in the medium using an LDH-Mix Cytotoxicity Test Kit, absorbance was measured at 490 nm (reading) and 720 nm (blank) with a spectrophotometer

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Lentiviral infection of cultured striatal neurons[1]
In vitro models of HD were realized using lentiviral vectors as described (27). These models involve the lentiviral-mediated overexpression of N-terminal 171 amino acid fragments of wild-type Htt (with 18 glutamine repeats, 18Q) or mHtt (with 82 glutamine repeats, 82Q) in striatal neuronal cultures. For lentiviral-mediated protein expression, cultures were infected 24 h after seeding. On Day 4, half of the medium was replaced with the fresh medium supplemented with selisistat in 2× concentration. Treatments with compound were performed once a week thereafter by adding fresh medium with compound at 1× concentration. The strong promoter constructs (high expression, 5–10 times endogenous) resulted in polyQ-dependent cell death within 2–4 weeks in vitro, as assessed by reduced NeuN-positivity and NeuN-positive cell numbers. Htt-N171-82Q- but not Htt-N171-18Q-exposed cells also develop intracellular Htt inclusions at 1–2 weeks (high expression) or 2–4 weeks (moderate expression) in vitro.

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HEK293 cell transfection and treatments[1]
HEK 293-T cells were grown in DMEM containing 10% FBS, 1% Penstrep, 1% G-Max at 37°C and 10% CO2. 8 × 105 cells were seeded on MW6 plates and after 24 h, cells were transfected with 2.5 µg of total plasmid DNA using Lipofectamine 2000 according to the manufacturer's instructions. Plasmids expressing GCN5 (NM_021078.1), p65 (NM_021975.3), human_SirT1 (NM_012238.4) were purchased from OriGene Technologies, plasmid expressing the Drosophila gene Sir2 cDNA (LD07439) was ordered from DGRC and cloned into a pcDNA vector. Four hours after transfection, the Opti-MEM medium was removed and selisistat was diluted to 0.1, 1 and 10 µm (DMSO 0.1%, v/v, as control) in the culture medium and added to the cells. At 24 h posttransfection, cells were collected and lysed in RIPA buffer (150 mm NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm Tris, pH 8.0) with protease and phosphatase inhibitors (Complete EDTA-free protease inhibitor cocktail, Roche and PhosSTOP inhibitor cocktail, Roche). Total lysates were clarified by centrifugation at 3000g for 5 min and the protein amount quantified by BCA according to the manufacturer's instructions.

Drosophila crosses[1]
To compare phenotypes of Htt-expressing animals in normal versus a Sir2-altered background, flies that were elav-Gal4[C155]; Sir2[17]/CyO were crossed to UAS-Httex1p Q93 homozygotes (line p463). To produce the homozygous deletion of Sir2 in an HD background, flies that were elav-Gal4[C155]; Sir2[17]/CyO were crossed to UAS-Httex1p Q93 that contained Sir2[17] and a second chromosome marker. Crosses were performed at 22.5°C. After eclosion, adult flies were reared at 25°C on standard cornmeal molasses medium for genetic studies or medium containing either 0.1% DMSO or the indicated concentration of selisistat (0.1–10 µm) for pharmacological studies. Fresh food was provided daily. Pseudopupil analysis was performed at 7 days as described. For longevity experiments, freshly enclosed virgins were aged in groups of 25–30 animals. Longevity was determined by counting the number of surviving animals to calculate percent survival, and flies were passed every 2–3 days. Climbing of aged 7-day-old flies was performed in polystyrene shell vials 9.5 cm in height with a diameter of 2.4 cm. Vials were placed in a holding box with the front and back open. A light box was placed behind the vials to improve visibility of flies. The flies were video recorded using an Exilim EX-FH20 camera with 40 f.p.s. The percentage of flies that climbed past the midpoint of the vial was calculated as a function of time after shakedown. Approximately 10–15 flies per vial were used for the climbing assay.

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Drug treatments[1]
Treatments were started at 4.5 weeks of age after mice had been tested at 3.5–4 weeks to establish baseline behavioral performance for all of the animals. Groups of 18 mice (9 per gender) were assigned to each R6/2 group. Mice were balanced across experimental groups by body weight, CAG repeats, date of birth and litter size before testing began. Mice were run in open field, rotarod and grip strength at 3.5–4 weeks and treatment designations were rebalanced before drug treatments were started to ensure that behavioral performance was initially similar between treatment groups. Additional data used for rebalancing the groups included rotarod fall time, total distance travelled and total rearing frequency in the open field and grip strength. Mice received daily (QD) oral gavage (PO; 10 ml/kg) of selisistat (5 and 20 mg/kg) or its vehicle (0.5% hydroxyl-propylmethylcellulose Methocel K4M Premium in sterile water; 0.5% HPMC). Suspensions were prepared weekly and aliquotted into amber vials (light sensitive) for daily dosing; powdered drug was stored in a desiccator at 4°C. Vehicle was prepared bimonthly and stored at 4°C. Each vial was vortexed prior to dosing and contained a small stir bar and remained on a stir plate during dosing. A satellite group of animals for pharmacokinetic assessments were dosed from 3 to 10 weeks of age with selisistat. Following the last dose, animals were terminated and trunk blood samples were collected from three mice per group at 0.25, 0.5, 1, 6 and 24 h postdose in heparin-coated tubes kept on wet ice until centrifugation at 2700 RPM at +4°C. The supernatant was removed and plasma stored at −80°C until analysis using an LC–MS/MS method with a lower limit of quantitation of 5 ng/ml. Pharmacokinetic parameter estimates were achieved using WinNonlin, v. 5.01.1.

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In Vivo Pharmacokinetic Analysis. [2]
C57bl/6J mice were dosed intravenously (iv) or by oral gavage with 10 mg/kg of compound 1 (selisistat) or 35 in phosphate-buffered saline containing 4% DMSO and 10% cyclodextrin. Plasma was collected at 5, 15, 30, 60, and 90 min and 2, 4, 6, 8, and 24 h after dosing. Samples were analyzed by LCMS at Absorption Systems. Plasma samples were prepared by solid-phase extraction in a 96-well plate format. A 50-μL aliquot of plasma was combined with 300 μL of 1% phosphoric acid spiked with an internal standard (warfarin at 50 ng/mL). Plasma samples were transferred to a Waters Oasis HLB 30 mg extraction plate, washed with 5% methanol/water, and eluted with acetonitrile. The elute was evaporated to dryness under N2 at 37 °C and redissolved in 20% aqueous acetonitrile.[2]
Samples (25 μL) were injected onto a Keystone Hypersil BDS C18, 30 × 2.1 mm, 3 μm column and eluted at 0.3 mL/min. A gradient of 2.5 mM NH4OH−formic acid (pH 3.5) to 2.5 mM NH4OH−formic acid in 90% acetonitrile was run over 3 min. Mass spectra were acquired using a PE Sciex API4000 with electrospray interface. Quantification was performed against calibration curves generated by spiking compound 1 or 35 into blank heparinized male C57bl/6J mouse plasma (0.3, 1, 3, 10, 30, 100, 300, 1000 ng/mL final concentration). Percent oral bioavailability was calculated from the ratio of the area under the curve up to the last quantifiable time point after oral and iv dosing, respectively. Terminal elimination half-life was calculated from the data obtained after iv dosing.

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Male ZDF rats (body weight 300 ± 25 g) were procured from Central Lab Animal Inc. The rats were kept at normal temperature (24 ± 0.5 °C), relative humidity (54–58%), and a 12 h dark and light cycle, under specific pathogen proof conditions. The rats were acclimated to the laboratory conditions for ten days before the start of the experiment. The HFD, comprising carbohydrates, fats (60%), proteins, minerals, fiber, and vitamins was obtained from Research Diets, Inc. and fed to the rats for 11 weeks to induce diabetes. EX-527 (selisistat)(5 μg/kg, twice weekly) was administered intraperitoneally (i.p.) to HFD-fed rats for ten weeks. The normal diet-fed rats received diets which were devoid of fats. Glucose levels were determined by using a glucometer. A rat with a glucose level of more than 300 mg/dL was considered as diabetic and used for further study. All the experimental ZDF rats were randomly distributed into three groups (n = 6). Rats were anesthetized after 21 weeks of treatment. The abdominal vein was used for blood collection and transferred into heparinized tubes. Serum was obtained following the centrifugation of blood at 2000× g for 10 min and transferred immediately at −80 °C for storage until further analysis. The major organs (liver) were collected and perfused with saline and stored at −80 °C for further analysis, as shown in Figure 1.[3]

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Dissolved in DMSO; ~5 μg/rat; Intracerebroventricular injection

Male Sprague-Dawley rats

[1]. A potent and selective Sirtuin 1 inhibitor alleviates pathology in multiple animal and cell models of Huntington's disease. Hum Mol Genet. 2014;23(11):2995-3007.

[2]. Discovery of indoles as potent and selective inhibitors of the deacetylase SIRT1 [published correction appears in J Med Chem. 2007 Mar 8;50(5):1086]. J Med Chem. 2005;48(25):8045-8054.

[3]. EX-527 Prevents the Progression of High-Fat Diet-Induced Hepatic Steatosis and Fibrosis by Upregulating SIRT4 in Zucker Rats. Cells. 2020 Apr 29;9(5):1101.

6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide is a member of the class of carbazoles that is 2,3,4,9-tetrahydro-1H-carbazole which is substituted at position 1 by an aminocarbohyl group and at position 6 by a chlorine. It is a member of carbazoles, a monocarboxylic acid amide and an organochlorine compound.
Selective inhibitor of SIRT1 that does not inhibit histone deacetylase (HDAC) or other sirtuin deacetylase family members (IC50 values are 98, 19600, 48700, > 100000 and > 100000 nM for SIRT1, SIRT2, SIRT3, HDAC and NADase respectively). Enhances p53 acetylation in response to DNA damaging agents.

C13H13CLN2O

248.7081

248.071

C, 62.78; H, 5.27; Cl, 14.25; N, 11.26; O, 6.43

49843-98-3

(S)-Selisistat;848193-68-0;(R)-Selisistat;848193-69-1

5113032

Off-white to light yellow solid powder

2.5

2

1

1

17

323

0

ClC1C([H])=C([H])C2=C(C=1[H])C1C([H])([H])C([H])([H])C([H])([H])C([H])(C(N([H])[H])=O)C=1N2[H]

FUZYTVDVLBBXDL-UHFFFAOYSA-N

InChI=1S/C13H13ClN2O/c14-7-4-5-11-10(6-7)8-2-1-3-9(13(15)17)12(8)16-11/h4-6,9,16H,1-3H2,(H2,15,17)

6-Chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide

Selisistat; EX 527; SEN 0014196; 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide; SEN0014196; SIRT1 Inhibitor III; EX527; SEN-0014196; SEN0014196; EX-527

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.05 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (10.05 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (10.05 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 1% DMSO+30% polyethylene glycol+1% Tween 80:14mg/mL

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