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Screening method and application of small molecular compound for targeted inhibition of vitamin K epoxide reductase

A small molecule compound, epoxide technology, applied in the field of molecular cell biology and biochemistry, can solve problems such as unclear VKOR inhibitors

Pending 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] At present, the small molecules that inhibit VKOR activity are all derivatives of coumarins or derivatives of phenindiones, but it is not clear whether there are other VKOR inhibitors with chemical structures different from these two types of compounds

Method used

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  • Screening method and application of small molecular compound for targeted inhibition of vitamin K epoxide reductase
  • Screening method and application of small molecular compound for targeted inhibition of vitamin K epoxide reductase
  • Screening method and application of small molecular compound for targeted inhibition of vitamin K epoxide reductase

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0067] 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:

[0068] 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

[0073] In this embodiment, the screening method for small molecule compounds targeted to inhibit vitamin K epoxide reductase comprises the following steps:

[0074] 1) Screening of small molecule compounds that target the inhibition of vitamin K cycle

[0075] 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:

[0076] 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.

[0077] b. The small molecule compounds in the Maybridge Hit Finder small molecule compoun...

Embodiment 2

[0121] In this example, the 14 small molecular compounds screened out in Example 1 are used as inhibitors targeting vitamin K epoxide reductase. The structural formulas of the 14 small molecular compounds are as follows:

[0122]

[0123]

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Abstract

The invention discloses a screening method and application of a small molecular compound for targeted inhibition of vitamin K epoxide reductase, and belongs to the technical field of molecular cell biology and biochemistry. By constructing a vitamin K circulation small molecule inhibitor screening system (namely a double-reporter-gene cell system), high-throughput screening can be rapidly performed on a drug small molecule library, and a small molecule compound for targeted inhibition of vitamin K circulation is obtained; furthermore, by detecting the inhibition effect of the screened small molecular compound on the activity of the VKOR protein, the small molecular compound capable of specifically inhibiting the activity of the VKOR protein can be obtained. By adopting the method, compounds of which the structures are different from those of coumarin derivatives and phenylindanedione derivatives are screened for the first time, can specifically inhibit the activity of VKOR protein, have potential application value of being developed into rodenticide and anticoagulant drugs, and are lead compounds for developing rodenticide and anticoagulant drugs.

Description

technical field [0001] The invention relates to a screening method and application of a small molecule compound targeting to inhibit vitamin K epoxide reductase, and belongs 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 modification process and...

Claims

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Application Information

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IPC IPC(8): G01N33/543C12Q1/26A61K31/277A61K31/137A61K31/433A61K31/37A61K31/17A61K31/4422A61K31/454A61K31/136A61K31/122A61K31/352A61P7/04A61P39/02
CPCG01N33/543C12Q1/26A61K31/277A61K31/137A61K31/433A61K31/37A61K31/17A61K31/4422A61K31/454A61K31/136A61K31/122A61K31/352A61P7/04A61P39/02
Inventor 沈国民刘浩沈滟刘红丽曹青高蒙李三强
Owner HENAN UNIV OF SCI & TECH
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