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Screening method and application of small molecular compound for targeted inhibition of vitamin K dependent gamma-glutamyl carboxylase

A technology of small molecule compounds and glutamyl carboxylase, which is applied in the fields of molecular cell biology and biochemistry, can solve the problems of unreported screening methods for small molecule compounds

Active Publication Date: 2020-06-05
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The key enzymes of the vitamin K cycle (VKOR and VKGC) are important targets for the development of anticoagulant drugs (or rodenticides), but no specificity has been reported yet Small molecule compounds targeting VKGC and screening methods thereof

Method used

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  • Screening method and application of small molecular compound for targeted inhibition of vitamin K dependent gamma-glutamyl carboxylase
  • Screening method and application of small molecular compound for targeted inhibition of vitamin K dependent gamma-glutamyl carboxylase
  • Screening method and application of small molecular compound for targeted inhibition of vitamin K dependent gamma-glutamyl carboxylase

Examples

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Effect test

preparation example Construction

[0069] The preparation method of the purified human VKOR protein used in the examples and test examples is as follows: the gene encoding human VKOR protein (Gene ID: 79001) is cloned into the expression vector pPICZ-B (invitrogen) of Pichia pastoris, and its C-terminus is sequentially connected PreScission protease cleavage site and GFP-HIS10 tag; protein expression and purification were performed according to the kit (Product No. K171001, Thermofisher Company) and literature (Ren F, Logeman BL, Zhang X, Liu Y, Thiele DJ, YuanP. X-ray structures of The high-affinity copper transporter Ctr1.NatCommun.2019,27;10(1):1386.doi:10.1038 / s41467-019-09376-7.) was carried out by the method of expressing membrane protein in Pichia pastoris. The purification steps are as follows:

[0070] 1) Yeast cells expressing human VKOR protein were crushed and dissolved in lysate (2% DDM, 150mM NaCl, 50mM Tris-HCl pH 8.0, 10μg / mL DNase I, 2mM PMSF protease inhibitor, solvent is water), Stir and ext...

Embodiment 1

[0075] In this embodiment, the screening method for small molecule compounds that target inhibition of vitamin K-dependent γ-glutamyl carboxylase comprises the following steps:

[0076] 1) Screening of small molecule compounds that target the inhibition of vitamin K cycle

[0077] The small molecular compound to be selected is added to the culture medium of the dual reporter gene cell system containing vitamin K for cultivation, and the small molecular compound targeted to inhibit the vitamin K cycle is screened out. The specific operation is as follows:

[0078] a. Transfect the 293TRex cell line (invitrogen) with a plasmid expressing both the FIX-Gla-PC fusion gene and the reporter gene (as shown in SEQ ID NO.5, synthesized by a biological company) (see Document 1), and screen with hygromycin The cell system stably expressing FIX-gla-PC and Metridia Luciferase dual reporter genes is named 293TRex-F9-Met.

[0079] b. The small molecule compounds in the Maybridge Hit Finder s...

Embodiment 2

[0126] In this example, the application of the small molecule compound HF13141-H5 screened in Example 1 and its structural analog HTS03341 as an inhibitor targeting vitamin K-dependent γ-glutamyl carboxylase, the small molecule compound HF13141- The structural formulas of H5 and its structural analog HTS03341 are as follows:

[0127] (HF13141-H5), (HTS03341).

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Abstract

The invention discloses a screening method and application of a small molecular compound for targeted inhibition of vitamin K dependent gamma-glutamyl carboxylase, and belongs to the technical field of molecular cell biology and biochemistry. By constructing a vitamin K circulating small molecule inhibitor screening system, a drug small molecule library can be rapidly subjected to high-throughputscreening, and a small molecule compound for targeted inhibition of vitamin K circulation is obtained; furthermore, the inhibition effect of the small molecular compound on the activity of the VKOR protein is detected; and the screened small molecular compound is identified by using a VKORC1 and VKORC1L1 gene double knockout cell line, so as to obtain the small molecular compound for targeted inhibition of vitamin K-dependent gamma-glutamyl carboxylase (VKGC). By adopting the method, a small molecular compound capable of specifically inhibiting vitamin K-dependent gamma-glutamyl carboxylase isscreened for the first time, and the small molecule can be used for researching biochemical characteristics of VKGC protein and has potential application value of being developed into rodenticide andanticoagulant drugs.

Description

technical field [0001] The invention relates to a screening method and application of a small molecular compound targeting at inhibiting vitamin K-dependent γ-glutamyl carboxylase, belonging to the technical fields of molecular cell biology and biochemistry. Background technique [0002] Vitamin K plays an important role in the normal coagulation of blood and is mainly used in the prevention of bleeding disorders in newborn infants, in the prevention of internal bleeding, and as an antidote to vitamin K antagonists. Vitamin K participates in coagulation by regulating the post-translational modification of some coagulation factors (such as coagulation factor II, coagulation factor VII, and coagulation factor IX) (such as figure 1 shown). The γ-carboxylation modification of glutamic acid residues in the Gla domain of vitamin K-dependent coagulation factors is a necessary condition for its coagulation function. Vitamin K plays a central role in this post-translational modific...

Claims

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Application Information

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IPC IPC(8): G01N33/543C12Q1/26A61K31/502A61P7/02A01N43/58A01P11/00
CPCG01N33/543C12Q1/26A61K31/502A61P7/02A01N43/58
Inventor 沈国民刘红丽刘浩沈滟曹青高蒙
Owner HENAN UNIV OF SCI & TECH
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