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].

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Inhibited Δ6 desaturase activity in rat liver microsomes (IC₅₀ = 0.7 μM), reducing conversion of [¹⁴C]-linoleic acid to γ-linolenic acid. No significant inhibition of Δ9 desaturase observed at 10 μM [1]
Inhibited Δ5 desaturase in HepG2 cells (IC₅₀ = 3.1 μM) and Δ6 desaturase (IC₅₀ = 0.6 μM) in a multiplexed cell assay measuring fatty acid conversion via LC-MS [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].

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Topical application (1 mg/ear) reduced arachidonic acid (AA)-induced mouse ear edema by 47% (p < 0.05) and suppressed leukotriene B₄ (LTB₄) production by 53% in inflamed tissues [1]

Rat liver microsomes were incubated with [¹⁴C]-linoleic acid (substrate), NADH, and test compounds at 37°C for 20 min. Reactions were stopped with methanol, and metabolites extracted for thin-layer chromatography (TLC) analysis. Radiolabeled products were quantified via scintillation counting to calculate desaturase activity [1]
Human HepG2 cell membranes were used to assess desaturase activity. Cells were treated with compounds, then incubated with isotope-labeled fatty acid precursors (e.g., dihomo-γ-linolenic acid for Δ5 assay). Lipid extracts were analyzed by LC-MS to quantify metabolite conversion rates [4]

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].

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HepG2 cells were seeded in 96-well plates, cultured for 24 h, then treated with compounds for 24 h. Cells were pulsed with stable isotope-labeled fatty acids (e.g., ¹³C₁₈-linoleic acid for Δ6 assay). Lipids were extracted, methylated, and analyzed by LC-MS/MS to determine desaturase inhibition [4]

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.

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Male ICR mice (20-25 g) were used for AA-induced ear edema. SC-26196 (1 mg/ear) or vehicle (acetone) was applied topically 30 min before AA (0.5 mg/ear). Ear thickness was measured at 1 h post-AA. For LTB₄ analysis, inflamed ear tissues were homogenized in ethanol, and LTB₄ quantified by radioimmunoassay [1]

[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 crucial for immune function. Limited evidence suggests that immune cell activation involves the synthesis of endogenous PUFAs, but the mechanisms remain unclear. To investigate this, we examined the metabolism of 18:3n-3 in resting and activated peripheral blood mononuclear cells (PBMCs) and Jurkat T-cell leukemia cells. We collected PBMCs from 34 male and female patients and incubated them with [1-13C]18:3n-3 with or without concanavalin A (Con A). In unstimulated PBMCs, the conversion of 18:3n-3 was undetectable; however, after stimulation, the conversion was significantly upregulated. The major products were 20:3n-3 and 20:4n-3, while 18:4n-3 was undetectable, suggesting that the process mainly proceeds through initial elongation and Δ8 desaturation. The amount of PUFA synthesized in Jurkat cells was 17.4 times that in PBMCs. The major products of 18:3n-3 transformation in Jurkat cells were 20:4n-3, 20:5n-3, and 22:5n-3. 13C enrichment of 18:4n-3 and 20:3n-3 indicated that their initial elongation and Δ6 desaturation occurred in parallel. The FADS2 inhibitor SC26196 reduced PBMC proliferation but had no effect on Jurkat cells, suggesting that PUFA synthesis is involved in the regulation of PBMC mitosis. Con. A stimulation increased the expression of FADS2, FADS1, ELOVL5, and ELOVL4 mRNA in PBMCs. A single transcript corresponding to the major FADS2 isoform FADS20001 was detected in both PBMCs and Jurkat cells. PBMC activation induced hypermethylation of a 470bp region in the FADS2 5' regulatory sequence. Compared to quiescent peripheral blood mononuclear cells (PBMCs), the degree of methylation in this region was lower in Jurkat cells. These findings suggest that the synthesis of polyunsaturated fatty acids (PUFAs) involved in initial elongation and Δ8 desaturation is involved in regulating PBMC proliferation and may be transcribedly regulated through alterations in DNA methylation. These processes are dysregulated in Jurkat cells. This is of great significance for understanding the regulation of mitosis in normal and transformed lymphocytes. [2] Objective: To determine whether conjugated linoleic acid (CLA)-induced reduction in body fat depends on the metabolism of CLA by Δ6 desaturase, cyclooxygenase, or lipoxygenase. Methods and Procedures: Mice were fed diets containing or without CLA, and Δ6 desaturase inhibitor (SC-26196), cyclooxygenase inhibitor (aspirin), or lipoxygenase inhibitor (nordihydroguaiacol (NDGA)) for 2 weeks. Body fat percentage, lean body mass, fat pad weight, liver weight, and fatty acid concentration were measured. The Δ6 desaturase index was calculated, and the concentrations of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) in adipose tissue were measured to verify enzyme inhibition. Results: Inhibition of Δ6 desaturase and cyclooxygenase was confirmed. CLA led to a reduction in body fat (P < 0.001). Δ6 desaturase inhibitors blocked this reduction (P = 0.08), and the dosage of these inhibitors decreased the calculated index (P < 0.05). Aspirin and NDGA had no effect on body fat and did not interact with CLA. Discussion: Inhibition of Δ6 desaturase prevented the reduction in body fat caused by CLA. Therefore, the desaturated metabolites of CLA may be involved in the anti-obesity effect of CLA. This effect of CLA does not appear to depend on cyclooxygenase. Since NDGA did not inhibit lipoxygenase activity, we cannot draw conclusions about its importance in mediating the anti-obesity effect of CLA. [3]

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We optimized a multiplex cell assay to simultaneously measure the activities of acyl-CoA elongase, Δ-5 desaturase (Δ5D), Δ-6 desaturase (Δ6D), and Δ-9 desaturase (Δ9D) in HepG2 cells using a (14)C-labeled tracer. HepG2 cells expressed only human stearoyl-CoA desaturase-1 isoenzyme (SCD1). Delta5 and Delta9 desaturase activities were measured by the efficient conversion of [1-(14)C]-eicosatetrienoic acid (C20:3, cis-8,11,14) to (14)C-arachidonic acid (C20:4, cis-5,8,11,14) and [1-(14)C]-stearic acid to (14)C-oleic acid (C18:1, cis-9), respectively. CP-74006 effectively inhibits Delta5D activity with an IC50 value of 20 nM. Through Delta6 desaturation and elongation, it accumulates (14)C-eicosatetraenoic acid (C20:4, cis-8,11,14,17) as the major metabolite and (14)C-eicosatetrienoic acid (C20:3, cis-11,14,17) as a minor metabolite, thus simplifying the metabolism of [1-(14)C]-α-linolenic acid (C18:3, cis-9,12,15). This simplified metabolite profile allows for the definition of Delta6D activity by comparing the Delta6D/elongation enzyme activity index of the (14)C-(C20:4/C18:3) ratio with the elongation enzyme activity index of the corresponding (14)C-(C20:3/C18:3) ratio. SC-26196 and cycloglutaric acid specifically inhibited Delta6D and Delta9D activities, respectively, with IC50 values of 0.1 μM and 0.9 μM. This medium-throughput cell assay provides an effective tool for identifying specific desaturase and elongase inhibitors. [4] SC-26196 is a selective Δ6 desaturase inhibitor that reduces the synthesis of pro-inflammatory eicosic acid compounds (such as LTB₄) by limiting the production of arachidonic acid (AA) from linoleic acid. It has demonstrated anti-inflammatory efficacy in vivo and does not inhibit cyclooxygenase [1] and can be used as a pharmacological tool for studying polyunsaturated fatty acid (PUFA) metabolism; its inhibition of Δ6/Δ5 desaturase alters the PUFA profile in immune cells, affecting proliferation and inflammatory responses [2][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.)