A prodrug form of A-771726
Leflunomide is an agonist of aryl hydrocarbon receptor (AHR) [1].?As an immunoregulatory agent, Leflunomide can be administrated orally. A771726 (teriflunomide) is an active drug formed non-enzymatically from leflunomide [2].
Leflunomide has been demonstrated to expand Tregs levels in peripheral blood?and stem cells subsets as well as to increase the migration of these cells?into the injured kidney. ?Leflunomide has been displayed to increase cells expressed IL-10 and reduce cells expressed IL-17- and IL-23 in ischemic-reperfused kidney [1].
Leflunomide has shown to significantly lower the lipid peroxidation in male Wistar albino rats of sepsis. Meanwhile, leflunomide successfully inhibited the protein carbonyl rise and NO rise?in bowel [2]. Leflunomide has shown to reduce cell proliferation, inhibit?tumor marker expression, as well as down-regulate neuroendocrine marker ASCL1 protein expression in a dose- and time-dependent manner in human MTC-TT cells [3].
参考文献:
[1] Baban B1,?Liu JY,?Mozaffari MS. Aryl hydrocarbon receptor agonist, leflunomide, protects the ischemic-reperfused kidney: role of Tregs and stem cells. Am J Physiol Regul Integr Comp Physiol.?2012 Dec;303(11):R1136-46.
[2] Ozturk E1,?Surucu M2,?Karaman A3,?Samdanc? E4,?Fadillioglu E5. Protective effect of leflunomide?against?oxidative intestinal injury in a rodent model of sepsis. J Surg Res.?2014 Apr;187(2):610-5.
[3] Alhefdhi A1,?Burke JF,?Redlich A,?Kunnimalaiyaan M,?Chen H. Leflunomide?suppresses growth in human medullary thyroid cancer cells. J Surg Res.?2013 Nov;185(1):212-6.
Kinase experiment: | DHODase activity is measured by the DCIP colorimetric assay. This is a coupled assay in which oxidation of DHO and subsequent reduction of ubiquinone are stoichiometrically equivalent to the reduction of DCIP. Reduction of DCIP is accompanied by a loss of absorbance at 610 nm (ε=21500 M/cm). The assay is performed in a 96-well microtiter plate at ambient temperature (ca. 25°C). Stock solutions of 10 mM leflunomide and A771726 are prepared in dimethyl sulfoxide (DMSO) and these are diluted with reaction buffer (100 mM Tris and 0.1 % Triton X-100, pH 8.0) to prepare working stocks of the inhibitors at varying concentrations. For each reaction, the well contained 10 nM DHODase, 68 μM DCIP, 0.16 mg/mL gelatin, the stated concentration of ubiquinone, 10 μL of an inhibitor working stock to give the stated final concentration, and reaction buffer. After a 5-min equilibration period, the reaction is initiated by addition of DHO to the stated final concentrations. The total volume of reaction mixture for each assay is 150 μL, and the final DMSO concentration is ≤ 0.01% (v/v). The reaction progress is followed by recording the loss of absorbance at 610 nm over a 10-min period (during which the velocity remained linear). Velocities are reported as the change in absorbance at 610 nm per minute, and each reported value is the average of three replicates. In experiments where the DHO or ubiquinone concentration is varied, the other substrate is held constant at 200 μM. To determine the inhibitor potency of leflunomide and A771726, the effects of varying concentrations of the two compounds on the initial velocity of the DHODase reaction is measured over a concentration range of 0.01?1.0 μM. In these experiments the DHO and ubiquinone concentrations are held constant at 200 and 100 μM, respectively. |
参考文献: [1]. Davis JP, et al. The immunosuppressive metabolite of leflunomide is a potent inhibitor of human dihydroorotate dehydrogenase. Biochemistry. 1996 Jan 30;35(4):1270-3. | |
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