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Application of dezocine in preparation of nicotinamide phosphoribosyltransferase inhibitor

A technology of phosphoribosyl and nicotinamide, applied in the field of anti-tumor drugs, can solve the problem of not achieving tumor suppressive effect and the like

Inactive Publication Date: 2020-11-17
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, adverse reactions such as thrombocytopenia and gastrointestinal toxicity, and the results of the current phase I clinical study show that the NAMPT inhibitors under test did not achieve the expected tumor suppression effect

Method used

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  • Application of dezocine in preparation of nicotinamide phosphoribosyltransferase inhibitor
  • Application of dezocine in preparation of nicotinamide phosphoribosyltransferase inhibitor
  • Application of dezocine in preparation of nicotinamide phosphoribosyltransferase inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] In vitro CNBr-activated Sepharose 4B beads pull down experiment

[0053] The reagent used in embodiment 1 is composed as follows:

[0054] Buffer I: 0.1M NaHCO 3 and 0.5M NaCl, pH 8.3.

[0055] Buffer II: 0.1M Tris-HCl, pH 8.0.

[0056] Buffer III: 0.1M acetic acid and 0.5M NaCl, pH 4.0.

[0057] Buffer IV: 0.1M Tris-HCl and 0.5M NaCl, pH 8.

[0058] Lysis buffer: 50mmol / L Tris (Tris, pH7.5), 5mmol / L ethylenediaminetetraacetic acid (EDTA), 150mmol / L NaCl, 1mmol / L dithiothreitol (DTT), 0.01% ethylphenyl polyethylene glycol (NinidetP-40), 0.02 mmol / L phenylmethylsulfonyl fluoride (PMSF), 4 μg / mL bovine serum albumin (BSA) and protease inhibitors.

[0059] Wash buffer: 50mmol / L tris (Tris, pH7.5), 5mmol / L ethylenediaminetetraacetic acid (EDTA), 150mmol / L NaCl, 1mmol / L dithiothreitol (DTT), 0.01% ethylphenyl polyethylene glycol and 0.02mmol / L phenylmethylsulfonyl fluoride (PMSF).

[0060] Example 1 There are three groups of experiments. The first group uses MDA-MB-23...

Embodiment 2

[0079] Liquid chromatography tandem mass spectrometry (LC-MS) analyzes the differential bands of Example 1

[0080] (1) Excise the Coomassie-stained spot on the polyacrylamide gel of the second group in Example 1, transfer it to a 1.5 mL microcentrifuge tube, and wash it twice with Mill-Q water. The blot was then destained with 100 μL of 50 mM ammonium bicarbonate / acetonitrile (1:1, v / v) and incubated by vortexing occasionally for 30 min depending on the staining intensity. The destained solution was then discarded, washed twice with 200 μL Mill-Q water, and 40 μL of 100% acetonitrile was added for 15 min to dry the gel spot in a Speedvac for 10 min. Finally, spot the gel in a minimum volume of 20 μg / mL sequencing-grade porcine trypsin or chymotrypsin (Promega, USA) in 25 mM NH 4 HCO 3 rehydrate and incubate overnight at 37°C. Transfer the supernatant to a 200 μL microcentrifuge tube and extract the gel once with extraction buffer (67% acetonitrile with 1% trifluoroacetic a...

Embodiment 3

[0085] Perform western blot verification on the pull down results

[0086] Specifically, the pulldown product of MDA-MB-231 cell lysate and dezocine-coupled agar gel beads 4B and the cell lysate of human breast ductal carcinoma cell BT549 and dezocine-coupled agar gel The pulldown products of beads 4B were subjected to western blot using NAMPT antibody as the primary antibody to verify that dezocine can directly bind to NAMPT. The operation of western blot is as follows:

[0087] (1) Preparation of separation gel: Take 10% separation gel as an example, add the ingredients in Table 3 to a 50mL centrifuge tube in turn, shake well, add to a glass plate, and then add 1mL of isopropanol to seal the liquid surface.

[0088] Table 3 10% separation gel preparation (20mL)

[0089]

[0090] (2) Prepare 5% concentrated gel: After the separation gel is solidified, pour off the isopropanol and dry it with filter paper, then prepare the concentrated gel according to Table 4, mix well a...

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PUM

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Abstract

The invention relates to an application of dezocine in preparation of a nicotinamide phosphoribosyltransferase inhibitor. Tests prove that dezocine can be directly combined with nicotinamide phosphoribosyltransferase to inhibit the activity of nicotinamide phosphoribosyltransferase, and can be used as an active ingredient of a nicotinamide phosphoribosyltransferase inhibitor.

Description

technical field [0001] The invention relates to the technical field of antitumor drugs, in particular to the application of dezocine in the preparation of nicotinamide phosphoribosyltransferase inhibitors. Background technique [0002] Nicotinamide phosphoribosyltransferase (NAMPT), also known as visfatin or pre-B cell clone enhancer factor (PBEF), can synthesize nicotinamide mononucleoside from the precursor substance nicotinamide (NAM) Nicotinamide mono-nucleotide (NMN), and then nicotinamide mononucleotide (NMN) is converted to nicotinamide adenine dinucleoside under the action of nicotinamide mono-nucleotide adenylyltransferase (nicotinamide mono-nucleotide adenylyltransferase, NMNAT) acid (nicotinamide adenine dinucleotide, NAD). Nicotinamide adenine dinucleotide (NAD) is one of the important coenzymes in cellular redox reactions and plays a vital role in various cellular physiological processes. For example, NAD is a substrate for sirtuins, poly ADP-ribose polymerase...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/135A61P35/00A61P43/00
CPCA61K31/135A61P35/00A61P43/00
Inventor 郑多薛晨阳王翰林王霆邹永东袁爱武
Owner SHENZHEN UNIV
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