Delta6 desaturase (D6D, FADS2) (IC50=0.2 μM)

The proliferation of peripheral blood mononuclear cells (PBMC) is inhibited by SC-26196 (200 nM), but not that of Jurkat cells [2].

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For technical reasons, [13C]18:3n-3 could not be used in the experiment to test the effect of SC-26196 on PUFA synthesis in PBMCs. Consequently, the results are presented as the proportion of total n-3 PUFA. SC26196 significantly reduced the proportion of 20:4n-3 in stimulated PBMCs which was accompanied by a non-significant trend (P = 0.07) towards an increase in the proportion of 20:3n-3. There were also non-significant trends (P < 0.01) towards lower proportions of 20:5n-3 and 22:5n-3 (Figure 2B). Treatment of Jurkat cells with SC-26196 (200 nmoles/l) significantly increased [13C] enrichment of 18:3n-3 (59%; P = 0.009) and of 20:3n-3 (2-fold; P = 0.001), and decreased enrichment of 20:4n-3 (30%; P = 0.04), 20:5n-3 (19%; P = 0.02), and 22:5n-3 (33%; P = 0.0004) (Figure 2D). There was no significant effect of SC26196 on [13C] enrichment of 18:4n-3 and 22:6n-3.[2]

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SC-26196 and sterculic acid specifically inhibit the Delta6D and Delta9D activities with an IC(50) value of 0.1 microM and 0.9 microM, respectively. This medium-throughput cell assay provides an efficient tool in the identification of specific desaturase and elongase inhibitors[4].

The computed Δ6-desaturase index in adipose tissue and liver decreased with the addition of SC-26196 to the diet (at doses of 0, 0.07, 0.21, or 0.7 mg/kg to obtain dosages of 0, 10, 30, and 100 mg/kg per day). Δ6-desaturase is inhibited when 100 mg of SC-26196 are fed daily per kilogram of body weight [3].

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Decreased synthesis of arachidonic acid by inhibition of the Delta6 or Delta5 desaturase was evaluated as a means to mitigate inflammation. Using quantitative in vitro and in vivo radioassays, novel compounds representing five classes of Delta5 desaturase inhibitors and one class of Delta6 desaturase inhibitor were identified. The Delta6 desaturase inhibitor, SC-26196, had pharmacokinetic and pharmacodynamic profiles in mice that allowed for the evaluation of the pharmacological effects of chronic inhibition of desaturase activity. SC-26196 decreased edema to the same extent as indomethacin or essential fatty acid deficiency in the carrageenan paw edema model in the mouse. The antiinflammatory properties of SC-26196 were consistent with its mechanism of action as a Delta6 desaturase inhibitor: 1) A correlation existed between inhibition of liver Delta6 desaturase activity and decreases in edema. 2) The onset of the decrease in edema was time dependent. 3) Selective reduction of arachidonic acid occurred dose dependently in liver, plasma and peritoneal cells. 4) In the presence of SC-26196, controlled refeeding of arachidonic acid, but not oleic acid, reversed the changes resulting from desaturase inhibition. The Delta6 desaturase may be a target for development of antiinflammatory drugs whose mechanism of action is unique [1].

Cell proliferation assay [2]
Cell Types: PBMC and Jurkat cells
Tested Concentrations: 200 nM
Incubation Duration: 96 hrs (hours) for PBMC; 144 hrs (hours) for Jurkat cells
Experimental Results: PBMC treatment Dramatically diminished the proportion of dividing cells, division index and proliferation index. Cell proliferation of Jurkat cells was not Dramatically altered.

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Measurement of Cell Proliferation [2]
Peripheral blood mononuclear cell proliferation was measured by the dye dilution method. Cryopreserved PBMCs were thawed and 40 × 106 viable cells were suspended in 1 ml PBS containing 5% (v/v) FBS. PBMCs were either untreated or stimulated with Con. A (final concentration 5 µg/ml) and maintained in a humidified cell culture incubator at 37°C in a 5% CO2 atmosphere. Cells were stained with carboxyfluorescein succinimidyl ester according to the manufacturer’s instructions. Proliferation of Jurkat cells was measured by cell counting. Cells were seeded at 5 × 105 cells/ml in RPMI-1640 medium containing 10% (v/v) FBS and incubated for up to 144 h either with or without SC-26196 (200 nM) or DMSO (final concentration 0.02% (v/v)). Aliquots were collected at 24 h intervals and cell number was determined using a Coulter Z1 Cell Counter [2].

Animal/Disease Models: Male mice (12 or 15 weeks old) [3]
Doses: 0, 10, 30 and 100 mg/kg daily
Doses: included in the diet at 0, 0.07, 0.21 or 0.7 mg/kg diet to achieve doses of 0, 10, 30 and 100 mg/kg per day.
Experimental Results: Caused a decrease in calculated Δ6-desaturase index in adipose tissue and liver.

[1]. Novel, selective delta6 or delta5 fatty acid desaturase inhibitors as antiinflammatory agents in mice. J Pharmacol Exp Ther. 1998 Oct;287(1):157-66.

[2]. Polyunsaturated Fatty Acid Biosynthesis Involving Δ8 Desaturation and Differential DNA Methylation of FADS2 Regulates Proliferation of Human Peripheral Blood Mononuclear Cells. Front Immunol. 2018 Mar 5;9:432.

[3]. Conjugated linoleic acid-induced fat loss dependence on Delta6-desaturase or cyclooxygenase. Obesity (Silver Spring). 2008 Oct;16(10):2245-52.

[4]. A multiplexed cell assay in HepG2 cells for the identification of delta-5, delta-6, and delta-9desaturase and elongase inhibitors. J Biomol Screen. 2010 Feb;15(2):169-76.

Polyunsaturated fatty acids (PUFAs) are important for immune function. Limited evidence indicates that immune cell activation involves endogenous PUFA synthesis, but this has not been characterised. To address this, we measured metabolism of 18:3n-3 in quiescent and activated peripheral blood mononuclear cells (PBMCs), and in Jurkat T cell leukaemia. PBMCs from men and women (n = 34) were incubated with [1-13C]18:3n-3 with or without Concanavalin A (Con. A). 18:3n-3 conversion was undetectable in unstimulated PBMCs, but up-regulated when stimulated. The main products were 20:3n-3 and 20:4n-3, while 18:4n-3 was undetectable, suggesting initial elongation and Δ8 desaturation. PUFA synthesis was 17.4-fold greater in Jurkat cells than PBMCs. The major products of 18:3n-3 conversion in Jurkat cells were 20:4n-3, 20:5n-3, and 22:5n-3. 13C Enrichment of 18:4n-3 and 20:3n-3 suggests parallel initial elongation and Δ6 desaturation. The FADS2 inhibitor SC26196 reduced PBMC, but not Jurkat cell, proliferation suggesting PUFA synthesis is involved in regulating mitosis in PBMCs. Con. A stimulation increased FADS2, FADS1, ELOVL5 and ELOVL4 mRNA expression in PBMCs. A single transcript corresponding to the major isoform of FADS2, FADS20001, was detected in PBMCs and Jurkat cells. PBMC activation induced hypermethylation of a 470bp region in the FADS2 5'-regulatory sequence. This region was hypomethylated in Jurkat cells compared to quiescent PBMCs. These findings show that PUFA synthesis involving initial elongation and Δ8 desaturation is involved in regulating PBMC proliferation and is regulated via transcription possibly by altered DNA methylation. These processes were dysregulated in Jurkat cells. This has implications for understanding the regulation of mitosis in normal and transformed lymphocytes.[2]

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Objective: To determine whether conjugated linoleic acid (CLA)-induced body fat loss is dependent upon metabolism of CLA by Delta6-desaturase, cyclooxygenase, or lipoxygenase. Methods and procedures: Mice were fed diets with or without CLA and inhibitors to either Delta6-desaturase (SC-26196), cyclooxygenase (aspirin), or lipoxygenase (nordihydroguaiaretic acid (NDGA)) for 2 weeks. Body fat percent, lean mass, fat pad weights, liver weight, and fatty acid concentrations were determined. A Delta6-desaturase index was calculated, and adipose tissue prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)) concentrations were determined to confirm enzyme inhibition. Results: Inhibition of Delta6-desaturase and cyclooxygenase were confirmed. CLA caused a loss of body fat (P < 0.001). The body fat loss was blocked (P = 0.08) by the Delta6-desaturase inhibitor at a dose that decreased (P < 0.05) the calculated index. Aspirin and NDGA had no effect on body fat and did not interact with CLA. Discussion: Inhibition of Delta6-desaturase prevented CLA from being able to cause a body fat loss. Therefore, a desaturated metabolite of CLA appears to be involved in the CLA antiobesity effect. This effect of CLA does not seem dependent upon cyclooxygenase. Because lipoxygenase activity was not blocked by NDGA, we cannot draw conclusions about its importance in mediating the antiobesity effect of CLA. [3]

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A multiplexed cell assay has been optimized to measure the activities of fatty acyl-CoA elongase, delta-5 desaturase (Delta5D), delta-6 desaturase (Delta6D), and delta-9 desaturase (Delta9D) together using (14)C-labeled tracers in HepG2 cells, which express the human stearoyl-CoA desaturase-1 isoform (SCD1) exclusively. The Delta5 and Delta9 desaturase activities are indexed by the efficient conversion of [1-(14)C]-eicosatrienoic acid (C20:3, cis-8,11,14) to (14)C-arachidonic acid (C20:4, cis-5,8,11,14) and the conversion of [1-(14)C]-stearic acid to (14)C-oleic acid (C18:1, cis-9), respectively. CP-74006 potently blocks the Delta5D activity with an IC(50) value of 20 nM and simplifies the metabolism of [1-(14)C]-alpha-linolenate (C18:3, cis-9,12,15) by accumulating (14)C-eicosatetraenoic acid (C20:4, cis-8,11,14,17) as the major (14)C-eicosatrienoic acid (C20:3, cis-11,14,17) and (14)C-docosatetraenoic acid (C22:4, cis-10,13,16,19) as the minor metabolites through Delta6 desaturation and elongation. This simplified metabolite spectrum enables the delineation of the Delta6D activity by comparing the combined Delta6D/elongase activity index of the (14)C-(C20:4/C18:3) ratio with the corresponding elongation index of the (14)C-(C20:3/C18:3) ratio following compound treatment. SC-26196 and sterculic acid specifically inhibit the Delta6D and Delta9D activities with an IC(50) value of 0.1 microM and 0.9 microM, respectively. This medium-throughput cell assay provides an efficient tool in the identification of specific desaturase and elongase inhibitors. [4]

C27H29N5

423.55266

423.242

C, 76.56; H, 6.90; N, 16.53

218136-59-5

9845201

Off-white to yellow solid powder

1.1±0.1 g/cm3

650.2±55.0 °C at 760 mmHg

347.0±31.5 °C

0.0±1.9 mmHg at 25°C

1.607

3.2

0

5

8

32

599

0

C1CN(CCN1CCCC(C#N)(C2=CC=CC=C2)C3=CC=CC=C3)/N=C/C4=CN=CC=C4

QFYKXKMYVYOUNJ-JBASAIQMSA-N

InChI=1S/C27H29N5/c28-23-27(25-10-3-1-4-11-25,26-12-5-2-6-13-26)14-8-16-31-17-19-32(20-18-31)30-22-24-9-7-15-29-21-24/h1-7,9-13,15,21-22H,8,14,16-20H2/b30-22+

2,2-diphenyl-5-[4-[(E)-pyridin-3-ylmethylideneamino]piperazin-1-yl]pentanenitrile

218136-59-5; SC-26196; (E)-2,2-Diphenyl-5-(4-((pyridin-3-ylmethylene)amino)piperazin-1-yl)pentanenitrile; 2,2-Diphenyl-5-(4-((pyridin-3-ylmethylene)amino)piperazin-1-yl)pentanenitrile; 2,2-diphenyl-5-[4-[(E)-pyridin-3-ylmethylideneamino]piperazin-1-yl]pentanenitrile; CHEMBL4554790; SCHEMBL20580676; CHEBI:232585;

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 : ~5 mg/mL (~11.80 mM)

Solubility in Formulation 1: 10 mg/mL (23.61 mM) in 15% Cremophor EL 85% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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: 5 mg/mL (11.80 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; Need ultrasonic and warming and heat to 40°C.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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