Method for preparing crosslinked NAMPT (Nicotinamide Phosphoribosyltransferase) and screening NAMPT inhibitor

An inhibitor, the technology of group A, applied in the direction of biochemical equipment and methods, microbiological measurement/testing, enzymes, etc., can solve the problems of low work efficiency, many reaction steps, complicated operation, etc., and achieve high work efficiency and reflection steps The effect of less and simple operation

Active Publication Date: 2014-07-02
WUHAN INST OF PHYSICS & MATHEMATICS CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] (1) The method of transforming the structure of the lead compound and the known inhibitor core compound to obtain the candidate inhibitor, and reacting the candidate inhibitor with the tumor cell line to obtain the finished product of the inhibitor. Only one candidate inhibitor can be treated at a time Screening, the steps are cumbersome, not only the cost is relatively high, but also

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  • Method for preparing crosslinked NAMPT (Nicotinamide Phosphoribosyltransferase) and screening NAMPT inhibitor
  • Method for preparing crosslinked NAMPT (Nicotinamide Phosphoribosyltransferase) and screening NAMPT inhibitor
  • Method for preparing crosslinked NAMPT (Nicotinamide Phosphoribosyltransferase) and screening NAMPT inhibitor

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preparation example Construction

[0051] The method for preparing cross-linked NAMPT and screening NAMPT inhibitors of the present invention comprises the following steps:

[0052] S1: Preparation of NAMPT recombinant protein.

[0053] S2: Add 1 part of NAMPT recombinant protein to 20-100 parts (preferably 30 parts) of reducing agent according to molar parts. The reducing agent can be selected from DTT (dithiothreitol) or TCEP (tris(2-carboxyethyl) ); incubate at a temperature of 20°C to 25°C for 0.5h to 1.5h (preferably 1h) to obtain crude protein, and use a desalting column to remove the reducing agent in the crude protein to obtain pretreated protein;

[0054] S3: Add 1 part of pretreated protein and 2 to 5 parts of cross-linking reagent BMB (1,4-bis(maleimido)butane) into the reaction kettle according to the molar parts, mix well, and put them at a temperature of 20℃~ After reacting for 1h-5h (preferably 2h) at 25°C, use a desalting column to remove unreacted BMB to obtain cross-linked NAMPT;

[0055] S4...

Embodiment 1

[0075] Example 1, the candidate inhibitor is selected as rosmarinic acid.

[0076] S1: Preparation of NAMPT recombinant protein.

[0077] S2: Add 2 mL of NAMPT recombinant protein with a concentration of 200 μM to 1.6 mL of DTT (dithiothreitol) with a concentration of 5 mM, and incubate for 1 h at a temperature of 20 ° C to obtain crude protein, and use a desalting column to remove the crude protein In the DTT, the pretreated protein was obtained.

[0078] S3: Add the pretreated protein and 80 μL of BMB cross-linking reagent with a concentration of 10 mM into the test tube, mix well, react at 20° C. for 2 hours, and remove excess BMB with a desalting column to obtain cross-linked NAMPT.

[0079] S4: preset PBS buffer solution, divide the PBS buffer solution into three parts: the first PBS buffer solution, the second PBS buffer solution and the third PBS buffer solution, wherein the volume of the first PBS buffer solution and the second PBS buffer solution Similarly, NAMPT re...

Embodiment 2

[0090] Example 2: Selection of the candidate inhibitor as cynarin.

[0091] S1: Preparation of NAMPT recombinant protein.

[0092] S2: Add 3 mL of NAMPT recombinant protein with a concentration of 200 μM to 36 mL of ECET with a concentration of 1 mM, incubate for 0.5 h at a temperature of 25°C to obtain crude protein, and use a desalting column to remove DTT in the crude protein to obtain pretreated protein .

[0093] S3: Add the pretreated protein and 240 μL of BMB with a concentration of 10 mM into the test tube, mix well, react at a temperature of 20° C. for 1 hour, and remove excess BMB with a desalting column to obtain cross-linked NAMPT.

[0094] S4: preset PBS buffer solution, divide the PBS buffer solution into three parts: the first PBS buffer solution, the second PBS buffer solution and the third PBS buffer solution, wherein the volume of the first PBS buffer solution and the second PBS buffer solution Similarly, NAMPT recombinant protein was added to the first PBS...

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Abstract

The invention discloses a method for preparing crosslinked NAMPT (Nicotinamide Phosphoribosyltransferase) and screening a NAMPT inhibitor and relates to the field of inhibitors. The method comprises the following steps: preparing a NAMPT recombinant protein; reducing the NAMPT recombinant protein with a reducing agent; carrying out a reaction between the reduced protein and a crosslinking agent so as to obtain the crosslinked NAMPT the active center of which is plugged; compounding a first protein solution and a second protein solution; preparing a first solution to be analyzed, a first contrast solution, a second solution to be analyzed and a second contrast solution; respectively measuring the fluorescence intensities of the first solution to be analyzed, the first contrast solution, the second solution to be analyzed and the second contrast solution by using a fluorescence spectrophotometer; and calculating the descending percentage of the fluorescence intensity of the first solution to be analyzed and the second solution to be analyzed. The method comprises few steps, is simple to operate and low in cost; and the external environment hardly influences a reaction process. Thus, the method is good in repeatability and the result is relatively accurate.

Description

technical field [0001] The invention relates to the field of NAMPT inhibitor selection, in particular to a method for preparing cross-linked NAMPT and screening NAMPT inhibitors. Background technique [0002] NAMPT (nicotinamidephosphoribosyltransferase, nicotinamide phosphoribosyltransferase), also known as PBEF (pre-B cell colony-enhancing factor, pre-B cell clone enhancer factor) or Visfatin (cisceral fat adipokine, visceral fat), is the NAD ( Nicotinamde adenine dinucleotide) is the rate-limiting enzyme in the salvage synthesis pathway. NAD is used to participate in cell material, energy metabolism, protein modification, DNA repair and other projects. When the human body is in a state of stress or develops a disease, the content of NAD in the human body will increase, and the content of NAMPT corresponding to NAD will also increase. [0003] NAMPT is widely distributed in the human body. NAMPT has different mechanisms of action in human cells (cytoplasm, nucleus and mit...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12Q1/48G01N21/64
CPCC12N9/1077C12Q1/48C12Y204/02012G01N21/6428
Inventor 董旭唐淳
Owner WUHAN INST OF PHYSICS & MATHEMATICS CHINESE ACADEMY OF SCI
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