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Active fluorescence detection method of anthropogenic silent information regulatory factor 6 (Sirtuin6)

A technology for regulating factors and silencing, applied in the field of pharmacy, can solve the problems of misdirection and the decrease in the affinity of substrate peptides and SIRT6, and achieve the effect of accurate screening effect, tight binding and low cost.

Inactive Publication Date: 2015-02-18
GUIYANG MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the coumarin that these methods rely on can only be attached to the C-terminus of the polypeptide, so that the long-chain fatty acylated lysine of the substrate, the SIRT6 recognition site, can only be located at the C-terminus, resulting in the interaction between the substrate polypeptide and SIRT6. On the other hand, the coumarin group may also bring some misleading to the subsequent drug screening [K. T. Howitz, et al. Nature2003, 425, 191 ; M. Kaeberlein, et al. J. Biol. Chem.2005, 280, 17038; M.T. Borra, et al. J. Biol. Chem.2005, 280, 17187; D. Beher, et al. Chem. Biol. Drug Des.2009, 74, 619; M, Pacholec, et al. J. Biol. Chem.2010, 285, 8340]

Method used

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  • Active fluorescence detection method of anthropogenic silent information regulatory factor 6 (Sirtuin6)
  • Active fluorescence detection method of anthropogenic silent information regulatory factor 6 (Sirtuin6)
  • Active fluorescence detection method of anthropogenic silent information regulatory factor 6 (Sirtuin6)

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Embodiment 1

[0027] Embodiment 1 of the present invention: Activity fluorescence detection method of human sirtuin 6:

[0028] Reagents and Instruments

[0029] All reagents were purchased from Aldrich or Acros company of analytical grade without further processing; VARIAN INOVA 400M Hz nuclear magnetic resonance instrument; liquid mass spectrometer was Shimadzu-LC20A and Thermo LCQ FLEET mass spectrometer, and the analytical column was Sprite TARGA C18 column (40 × 2.1 mm, 5 μm, Higgins Analytical, Inc.), the detection wavelength is 215 and 280 nanometers, using the binary gradient elution method of 0.1% formic acid solution and 0.1% formic acid acetonitrile solution; BIO-TEK? Synergy H4 multifunctional enzyme label instrument (excitation wavelength 336 nm, emission wavelength 490 nm).

[0030] Synthesis and purification of peptides

[0031] Synthetic route such as image 3 As shown, long-chain fatty acylated peptides and other peptides were synthesized by Fmoc-Wang resin using sta...

Embodiment 2

[0054] Embodiment 2 of the present invention: the screening method of Sirtuin 6 regulator:

[0055] (Dabcyl)ISGASEK(Myristoyl)DIVHSE(Edans)G Screening for Inhibitors of SIRT6

[0056] The steps here are the same as those described above (fluorescence experiment of (Dabcyl)ISGASEK(Succinyl)DIVHSE(Edans)G polypeptide under the catalysis of SIRT6), except that different compounds to be tested (both at a concentration of 30uM) were added to the reaction. In the first step In the reaction, SIRT6 was added at the end to start the reaction. Experimental results such as Figure 7 shown.

[0057] Determination of IC50 of SIRT6 inhibitors

[0058] The steps here are the same as those described above ((Dabcyl)ISGASEK(Succinyl)DIVHSE(Edans)G screening SIRT6 inhibitors), except that different concentrations of Nicotinamide (50, 100, 200, 500, 1000, 2000uM). Experimental results such as Figure 8 shown.

[0059] according to Figure 7 It was learned that no SIRT6 and only SIRT6 ...

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Abstract

The invention discloses an active fluorescence detection method of an anthropogenic silent information regulatory factor 6 (Sirtuin6). A polypeptide containing fluorophore and quenching group (FRET (fluorescence resonance energy transfer) effect) and long-chain fatty acylation lysine residue is used as a substrate, the fluorescence intensity is associated with the enzymatic activity of Sirtuin6, and the Sirtuin6 recognition site (long-chain fatty acylation lysine) of the substrate is located in the middle of the substrate by the FRET effect, thus, the substrate (polypeptide) is close to a natural substrate of Sirtuin6, and the combination is close; more fluorophores can be selected, and reliable screening result can be obtained. The method can be applied to screening of the Sirtuin6 regulator or is used for active detection of Sirtuin6 in a biological sample, has the characteristics of miniaturization, automation, reliability, rapidness and the like, is suitable for high-reflux application, and also can be used for development of the novel Sirtuin6 active detection kit and screening of the Sirtuin6 regulator.

Description

technical field [0001] The invention relates to the field of pharmacy, in particular to an activity fluorescence detection method of human silent information regulator 6 (Sirtuin 6 or SIRT6). Background technique [0002] Histone deacetylases are a family of proteases that catalyze the hydrolysis of acetyl groups of the side chains of lysine residues in various substrate proteins. The human-derived histone deacetylases discovered so far include 18 subspecies in 4 subclasses. According to different catalytic mechanisms, they are divided into classic class I, class II and class IV histone deacetylases (HDACs, 11 types are HDAC1 to HDAC11) [A. J. de Ruijter, et al. Biochem. J. 2003, 370 , 737]; In addition, the relatively special class III histone deacetylase with nicotinamide adenine dinucleotide (NAD) as the coenzyme is also called silent information regulator (Sirtuin, there are 7 kinds of SIRT1 to SIRT7 ). According to the similarity of protein sequence and evolution c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 何彬李燕刘亭李勇军兰燕宇王爱民王永林
Owner GUIYANG MEDICAL UNIVERSITY
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