SIRT1

(S)-Selisistat is a SIRT1 enzymatic activity inhibitor (IC50, 98 nM) that was found via a high-throughput screen employing human SIRT1 expressed in bacteria. With an IC50 of 38 nM, (S)-Selisistat inhibits SIRT1 in a concentration-dependent manner that is comparable with the activity of SIRT1 expressed in bacteria. (S)-Selisistat does not inhibit class I/II HDAC activity at concentrations up to 100 μM, but it is substantially less effective against SIRT2 (IC50, 19.6 μM) or SIRT3 (IC50, 48.7 μM) [1]. SIRT1 activity is inhibited by (S)-Selisistat, while SIRT1 mRNA and protein levels are unaffected [2].

In ob/ob septic mice, (S)-Selisistat eliminates the mitigating effect of resveratrol (RSV)-induced microvascular inflammation. Lastly, compared to the sepsis+vehicle group, the 7-day survival rate of ob/ob mice in the sepsis+RSV group was considerably higher [3].

Class I and II HDAC Fluorimetric Assay. [4]
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. [4]
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. [4]
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. [4]
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.

In Vivo Pharmacokinetic Analysis. [4]
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.

Pharmacokinetic Results [Br J Clin Pharmacol. 2015 Mar;79(3):477-91.]
In male subjects, after a single oral dose of 5 to 600 mg of selisistat in a fasting state, the drug was rapidly absorbed, but the absorption rate appeared to be dose-dependent, with the median time to peak concentration (tmax) of selisistat increasing from 1 hour after administration in the 5 mg dose group to 4 hours after administration in the 600 mg dose group (Figure 1). The elimination of selisistat was biphasic, with the apparent terminal plasma half-life appearing to increase with increasing dose (mean range from 1.6 hours in the 5 mg dose group to 6.1 hours in the 600 mg dose group). The AUC(0,∞) of selisistat increased dose-proportionately in the dose range of 5 to 300 mg, and the increase in AUC(0,∞) was significantly greater than that at dose levels between 300 and 600 mg dose levels, suggesting that one or more clearance mechanisms approach saturation at high doses (Figure 2A and Table 2). In all male subjects, the urinary excretion of the unchanged drug was low, with less than 0.02% excreted within 24 hours after administration at all dose levels. Following multiple administrations, the urinary excretion of the unchanged drug remained low but increased over time, consistent with observed plasma accumulation. Food had minimal effect on the pharmacokinetics of a single dose of selisistat in male subjects. A high-fat breakfast delayed the rate of absorption, but the extent of absorption remained essentially unchanged. Pharmacokinetic studies of selisistat with multiple oral administrations showed no dose- or time-dependent effects on tmax and apparent terminal half-life. At each dose level, the morning trough plasma concentration of selisistat typically reached steady state on day 4. Consistent with single-dose results, steady-state AUC(0,τ) increased proportionally over a once-daily dose range of 100 mg to 300 mg (Figure 2B), while steady-state Cmax increased proportionally to the dose. Furthermore, the steady-state AUC(0,τ) of the twice-daily 100 mg dose group was approximately twice that of the once-daily 100 mg dose group (Table 3). In the single-dose phase, the coefficients of variation (%CV) for AUC(0,∞) and Cmax between subjects were 35%–71% and 23%–46%, respectively. At all dose levels, the pooled inter-subject variability for AUC(0,∞) and Cmax was 56% and 33%, respectively. In the multiple-dose phase, the inter-subject variability (%CV) was 17%–59% for male subjects and 28%–68% for female subjects. After both single and multiple doses, systemic exposure was higher in women than in men. AUC(0,∞), AUC(0,τ), and Cmax values were 1.1-fold, 2.2–2.3-fold, and 1.7–1.9-fold higher in female subjects than in male subjects, respectively. There were no differences in systemic exposure or pharmacokinetic parameter estimates between Caucasian and non-Caucasian subjects.

Safety [Br J Clin Pharmacol. 2015 Mar;79(3):477-91.]
No serious adverse events were reported during the study, and no subjects withdrew from the study due to adverse events. Single oral doses of selisistat up to 600 mg were considered safe and well-tolerated in healthy male subjects, and 300 mg in female subjects (Table 5). Single oral doses of selisistat up to 300 mg once daily for 7 days were also considered safe and well-tolerated in healthy male subjects, and 100 mg twice daily for 7 days in healthy female subjects. The incidence of drug-related adverse events was low in male subjects, and the number of subjects experiencing adverse events did not increase with increasing selisistat dose. The incidence of adverse events did not exceed that in the placebo group. No increase in the number of adverse event reports was observed after multiple doses of celisstat compared to a single dose (Table 6). Most adverse events reported by both male and female subjects were mild and resolved without treatment. Only one serious adverse event occurred during the study period. An 18-year-old male subject experienced orthostatic syncope 1 hour and 18 minutes after taking 150 mg of celisstat. The investigators considered this event to be likely related to the study drug. Dietary status had no effect on adverse events. The most common drug-related adverse event after a single oral dose of celisstat was headache, occurring in 12% of male subjects and 83% of female subjects. The incidence of adverse events was lower in male subjects after multiple oral doses of celisstat. Among female subjects, 3 out of 6 subjects reported at least one gastrointestinal upset. Overall, the frequency of adverse event reports was higher in women taking the drug and in men taking placebo (Tables 5 and 6). No dose- or treatment-related trends were found in clinical laboratory assessments (including liver function tests, hematological parameters, vital signs, or cardiac function). Specifically, no treatment- or dose-related trends were observed in the parameters recorded by the 12-lead safety ECG, and no clinically significant abnormalities were found in the ECG morphology at any dose level of celisilatal treatment. Based on the 12-lead safety ECG assessment, no subjects had a QTc interval >480 ms or an increase >60 ms from baseline. No clinically significant abnormalities were found in physical examination, postural control, or neurological examination, and the rocking platform test performance remained unchanged.

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Concentration-effect model of ECG parameters [Br J Clin Pharmacol. 2015 Mar; 79(3):477-91.]
QTc data showed low variability as measured by the standard deviation of ΔQTcF between subjects, with 5.3 ms for the single-increment (SAD) portion and 6.8 ms for the multiple-increment (MAD) portion. Table 7 shows the changes in QTcF relative to baseline for each dose group in Part 1 (highest plasma concentration). Patterns at various time points and dose groups indicated that selisistat had no dose-dependent effect on the QTc interval. Within the observed plasma concentration range, no significant concentration-dependent effect of ΔΔQTcF was observed after a single administration of 5 mg to 600 mg selisistat. Linear models with intercepts fitted the data well, with estimated overall intercepts and slopes of 0.9 ms (90% CI −0.2, 2.0) and −0.00026 ms/ng ml−1 (90% CI −0.00063, 0.00010), respectively (Figure 3A). Similar results were obtained from the analysis of the MAD portion of the data, with an intercept of 2.8 ms (90% CI −0.16, 5.71) and an estimated slope of −0.00011 ms/ng ml−1 (90% CI −0.00087, 0.00066; Figure 3B). Using this model, the predicted ΔΔQTcF effect after a single 600 mg dose, at an observed geometric Cmax of 26.6 μm, was −0.9 ms (90% CI −3.3, 1.4). After a once-daily 300 mg dose for 7 consecutive days, with an observed Cmax of 22.5 μM, the predicted ΔΔQTcF effect was approximately 2.8 ms (90% CI −0.1, 5.6). For plasma concentrations exceeding the mean Cmax (e.g., 30 μM), using the same model, the predicted QTcF effect was 3.7 ms (90% CI −0.1, 7.5). In both the single-dose (SAD) and multiple-dose (MAD) portions of the study, the upper limit of the 90% CI for the predicted ΔΔQTcF effect was less than 10 ms at all observed plasma concentrations (Figures 3A and 3B).

[1]. Inhibition of SIRT1 catalytic activity increases p53 acetylation but does not alter cell survival following DNA damage. Mol Cell Biol. 2006 Jan;26(1):28-38.

[2]. SIRT1 is a regulator in high glucose-induced inflammatory response in RAW264.7 cells. PLoS One. 2015 Mar 20;10(3):e0120849.

[3]. Resveratrol attenuates microvascular inflammation in sepsis via SIRT-1-Induced modulation of adhesion molecules in ob/ob mice. Obesity (Silver Spring). 2015 Jun;23(6):1209-17.

[4]. Discovery of indoles as potent and selective inhibitors of the deacetylase SIRT1. J Med Chem. 2005 Dec 15;48(25):8045-54.

(S)-selisistat is a 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide with the S configuration, which is its active enantiomer. It is a Sir1 inhibitor and is the enantiomer of (R)-selisistat. Human SIRT1 is an enzyme capable of deacetyling p53, a tumor suppressor protein, and is thought to regulate p53-dependent functions, including DNA damage-induced cell death. In this study, we used a novel, highly efficient, and specific small-molecule SIRT1 catalytic activity inhibitor, EX-527, to investigate the role of SIRT1 in p53 acetylation and cell survival after DNA damage. In primary human mammary epithelial cells and various cell lines, EX-527 treatment significantly increased the acetylation level of p53 lysine 382 after different types of DNA damage. It is noteworthy that the inhibition of SIRT1 catalytic activity by EX-527 had no effect on the growth, viability, or p53-regulated gene expression of cells treated with etoposide. Trichostatin A (TSA), a class I/II inhibitor of histone deacetylases (HDACs), also increased the level of acetylated p53. The synergistic effect of EX-527 and TSA increased the level of acetylated p53, confirming that p53 acetylation is co-regulated by SIRT1 and HDACs. Although TSA alone reduced cell survival after DNA damage, the combined use of EX-527 and TSA had no further effect on cell viability and growth. These results suggest that although SIRT1 can deacetylate p53, this effect does not affect cell survival after DNA damage in some cell lines and primary human mammary epithelial cells. [1] Sepsis is defined as a systemic inflammatory response syndrome that disrupts the function of the host immune system, including an imbalance between pro-inflammatory and anti-inflammatory responses mediated by immune macrophages. Sepsis can also induce acute hyperglycemia. Studies have shown that Silent Mating Signaling Regulator 2 Homolog 1 (SIRT1) is an NAD+-dependent deacetylase that mediates the deacetylation of NF-κB and inhibits its function. Therefore, SIRT1 may play an important role in the hyperglycemic-mediated inflammatory signaling pathway. This study demonstrates that hyperglycemia significantly downregulates the mRNA and protein levels of SIRT1 in RAW264.7 macrophages and upregulates the mRNA levels and release of two pro-inflammatory cytokines, IL-1β and TNF-α. Interestingly, the hyperglycemic-induced decrease in SIRT1 levels can be significantly upregulated by the SIRT1 activator SRT1720, while the levels and release of IL-1β and TNF-α are also significantly reduced. However, when SIRT1 function is inhibited using EX527 or its expression is suppressed using RNAi, the upregulation of IL-1β and TNF-α levels and release induced by hyperglycemia is further enhanced. In summary, these findings collectively indicate that SIRT1 is an important regulator of many hyperglycemic-related inflammatory diseases, such as sepsis. [2]

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Objective: Obesity is a SIRT1 deficiency state that increases morbidity and resource consumption in critically ill patients. SIRT-1 deficiency exacerbates microvascular inflammation and mortality in the early stages of sepsis. This study aimed to investigate the effects of the SIRT-1 activator resveratrol (RSV) on microvascular inflammation in obese septic mice. Methods: ob/ob and C57Bl/6 (wild-type) mice were pretreated with RSV or dimethyl sulfoxide (DMSO) (solvent control) before cecal ligation and puncture (sepsis). We investigated (1) leukocyte/platelet adhesion, (2) expression of E-selectin, ICAM-1 and SIRT-1 in the small intestine and (3) 7-day survival. A group of RSV-treated mice were treated with the SIRT-1 inhibitor (EX-527) after sepsis induction, and leukocyte/platelet adhesion and E-selectin/ICAM-1 expression were investigated. We treated human umbilical vein endothelial cells (HUVECs) with resveratrol (RSV) to investigate changes in E-selectin/ICAM-1 and p65 acetylation (AC-p65) expression under lipopolysaccharide (LPS) stimulation. Results: RSV treatment reduced leukocyte/platelet adhesion and E-selectin/ICAM-1 expression in septic ob/ob and wild-type mice, and increased SIRT-1 expression. RSV treatment reduced E-selectin/ICAM-1 expression by increasing SIRT-1 expression and decreased AC-p65 expression in HUVECs. EX-527 eliminated the RSV-induced reduction of microvascular inflammation in ob/ob septic mice. Finally, the 7-day survival rate of ob/ob mice in the sepsis + RSV group was significantly higher than that in the sepsis + vector group. Conclusion: RSV reduces microvascular inflammation and improves survival rate in ob/ob septic mice by increasing SIRT-1 expression. [3]

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High-throughput screening of human sirtuin SIRT1 has revealed a series of indole compounds that act as potent inhibitors with selectivity for SIRT1, superior to other deacetylases and NAD processing enzymes. The most active compound described in this paper has an IC50 value of 60-100 nM for SIRT1, which is 500 times higher than previously reported SIRT inhibitors. Enantiomerically pure indole derivatives were prepared to enable in vitro and in vivo characterization. Kinetic analysis showed that these inhibitors bind after nicotinamide is released from the enzyme and prevent the release of deacetylated peptides and O-acetyl-ADP-ribose (products of enzyme-catalyzed deacetylation). These SIRT1 inhibitors are small in molecular weight, highly permeable to cells, have high oral bioavailability, and are metabolically stable. These compounds provide chemical tools for studying the biological properties of SIRT1 and exploring the therapeutic uses of SIRT1 inhibitors. [4]

C13H13CLN2O

248.71

248.071

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

848193-68-0

Selisistat;49843-98-3;(R)-Selisistat;848193-69-1

707029

Off-white to light yellow solid powder

1.4±0.1 g/cm3

531.7±38.0 °C at 760 mmHg

275.4±26.8 °C

0.0±1.4 mmHg at 25°C

1.688

2.22

2

1

1

17

323

1

C1C[C@@H](C2=C(C1)C3=C(N2)C=CC(=C3)Cl)C(=O)N

FUZYTVDVLBBXDL-VIFPVBQESA-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)/t9-/m0/s1

(1S)-6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide

EX 527(S); 848193-68-0; (S)-selisistat; (1S)-6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide; (S)-6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide; Selisistat, (S)-; Selisistat S-enantiomer; MUD9R3TJV3; EX-527(S);(S)-Selisistat EX-527(S)

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)

DMSO : ≥ 100 mg/mL (~402.07 mM)

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