CN111239386B

CN111239386B - Screening method and application of small molecular compound for targeted inhibition of vitamin K-dependent gamma-glutamyl carboxylase

Info

Publication number: CN111239386B
Application number: CN202010067614.7A
Authority: CN
Inventors: 沈国民; 刘红丽; 刘浩; 沈滟; 曹青; 高蒙
Original assignee: Henan University of Science and Technology
Current assignee: Henan University of Science and Technology
Priority date: 2020-01-20
Filing date: 2020-01-20
Publication date: 2023-03-14
Anticipated expiration: 2040-01-20
Also published as: CN111239386A

Abstract

The invention discloses a screening method and application of a small molecular compound for targeted inhibition of vitamin K-dependent gamma-glutamyl carboxylase, belonging to the technical field of molecular cell biology and biochemistry. According to the invention, a vitamin K circulating small molecule inhibitor screening system is constructed, so that a drug small molecule library can be rapidly subjected to high-flux screening, and a small molecule compound for targeted inhibition of vitamin K circulation is obtained; further detecting the inhibition effect of the small molecule compound on VKOR protein activity, and identifying the screened small molecule compound by using VKORC1 and VKORC1L1 gene double-knockout cell lines to obtain the small molecule compound for targeted inhibition of vitamin K-dependent gamma-glutamyl carboxylase (VKGC). By adopting the method, a micromolecular compound for specifically inhibiting the vitamin K-dependent gamma-glutamyl carboxylase is screened for the first time, and the micromolecular compound can be used for researching the biochemical characteristics of VKGC protein and has potential application value in developing raticide and anticoagulant drugs.

Description

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

Technical Field

The invention relates to a screening method and application of a small molecular compound for targeted inhibition of vitamin K-dependent gamma-glutamyl carboxylase, belonging to the technical field of molecular cell biology and biochemistry.

Background

Vitamin K plays an important role in the normal blood coagulation and is mainly used for preventing the hemorrhagic diseases of newborn infants and preventing internal diseasesBleeding and antidotes to vitamin K antagonists. Vitamin K is involved in blood coagulation by modulating the post-translational modification of several coagulation factors (e.g., factor II, factor VII, factor IX) (as shown in FIG. 1). Gamma-carboxylation modification of glutamic acid residues in the Gla domain of vitamin K-dependent coagulation factors is a necessary condition for them to exert a coagulation function. Vitamin K plays a central role in this post-translational modification process and circulates in the body in three forms, vitamin K epoxide (KO), vitamin K (K) and dihydrovitamin K (KH) ₂ )。

Two enzymes are currently known to play a role in the vitamin K cycle. Wherein the vitamin K-dependent gamma-glutamyl carboxylase (VKGC) utilizes KH ₂ As a cofactor, glutamic acid residues of Gla domain of vitamin K-dependent coagulation factor are gamma-carboxylated and KH is simultaneously modified ₂ Conversion to KO. In order to allow the catalytic reaction of VKGC to continue, KO must be reduced to KH ₂ This reaction is mainly catalyzed by vitamin K epoxide reductase (VKOR) having KO reductase activity and K reductase activity. VKOR catalyzes KO to K, and K to KH ₂ So that the vitamin K can be recycled. Research shows that VKOR is a specific action target of a clinical anticoagulant warfarin (warfarin), and the warfarin inhibits the activity of VKOR to prevent KO from being converted into K and KH ₂ Thereby blocking the circulation of vitamin K, further inhibiting the gamma carboxylation modification of glutamic acid residue of the Gla structural domain of the blood coagulation factor and achieving the aim of anticoagulation. In addition, the raticide bromadiolone is also an inhibitor specifically targeting VKOR.

Key enzymes (VKOR and VKGC) of vitamin K circulation are important targets for developing anticoagulant drugs (or rodenticides), however, small molecular compounds specifically targeting VKGC and screening methods thereof are not reported at present.

Disclosure of Invention

The invention aims to provide a method for screening a small-molecule compound for targeted inhibition of vitamin K-dependent gamma-glutamyl carboxylase.

Meanwhile, the invention also provides application of the small molecular compound screened by the method in preparation of an inhibitor of the targeted vitamin K-dependent gamma-glutamyl carboxylase.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

a screening method of a small molecule compound for targeted inhibition of vitamin K-dependent gamma-glutamyl carboxylase comprises the following steps:

1) Screening of small molecule compounds for targeted inhibition of vitamin K cycle

Adding the micromolecule compound to be selected into a culture solution of a double-reporter gene cell system containing vitamin K for culturing, and screening out the micromolecule compound which can inhibit the circulation of the vitamin K in a targeted manner;

the double-reporter gene cell system contains a plasmid which can simultaneously express a FIX-Gla-PC fusion gene and a reporter gene, and the nucleotide sequence of the FIX-Gla-PC fusion gene is shown as SEQ ID NO. 1;

2) Screening small molecule compound without inhibiting VKOR protein activity

Carrying out VKOR protein activity inhibition determination on the screened small molecular compound for targeted vitamin K cycle inhibition, and screening the small molecular compound without VKOR protein activity inhibition;

3) Screening of small molecule compounds for targeted inhibition of vitamin K-dependent gamma-glutamyl carboxylase

Identifying the screened small molecular compound by using a cell line which has double knockout of VKORC1 and VKORC1L1 genes and simultaneously expresses a FIX-Gla-PC fusion gene to obtain the small molecular compound for targeted inhibition of vitamin K dependent gamma-glutamyl carboxylase.

According to the invention, a vitamin K circulating micromolecule inhibitor screening system (namely a double-reporter gene cell system) is constructed, so that a drug micromolecule library can be rapidly screened in a high flux manner, and a micromolecule compound for targeted inhibition of vitamin K circulation is obtained; further detecting the inhibition effect of the screened small molecular compounds on the activity of VKOR protein, screening small molecular compounds without the inhibition effect on the activity of VKOR protein; finally, identifying the screened small molecular compound by using a cell line which has double knockout of VKORC1 and VKORC1L1 genes and simultaneously expresses a FIX-Gla-PC fusion gene, and obtaining the small molecular compound for targeted inhibition of vitamin K dependent gamma-glutamyl carboxylase (VKGC). By adopting the method, a micromolecule compound which specifically inhibits the vitamin K dependent gamma-glutamyl carboxylase is screened for the first time, can be used for researching the biochemical characteristics of VKGC protein, has potential application value for developing rodenticide and anticoagulant drugs, and is a lead compound for developing the rodenticide and the anticoagulant drugs.

In the step 1), the host cell of the double-reporter gene cell system is human embryonic kidney cell 293Trex (invitrogen).

In the step 1), the amino acid sequence of the protein coded by the FIX-Gla-PC fusion gene (recombinant genes of humanized F9 and PROC genes) is shown as SEQ ID NO. 2.

In step 1), the reporter gene is a luciferase reporter gene, such as a beetle (firefly) luciferase reporter gene. The nucleotide sequence of the beetle luciferase reporter gene is shown as SEQ ID NO.3, and the amino acid sequence of the protein coded by the gene is shown as SEQ ID NO. 4.

Further preferably, in the step 1), the nucleotide sequence of the plasmid simultaneously expressing the FIX-Gla-PC fusion gene and the reporter gene (beetle luciferase reporter gene) is shown as SEQ ID NO. 5.

In the step 1), the culture medium of the double-reporter gene cell system culture solution is a complete culture medium containing double antibodies, and the double antibodies are penicillin and streptomycin respectively. The complete medium containing the double antibody comprises the following components: 10% FBS (fetal bovine serum, v/v), penicillin 100IU/mL, streptomycin 100. Mu.g/mL, DMEM (cat # SH30243.01, hyclone).

In the step 1), the concentration of vitamin K in the culture solution of the double-reporter gene cell system is 1-20 mu mol/L; more preferably 5. Mu. Mol/L.

In step 2), VKOR protein activity can be determined by conventional microsomal methods (see literature: fasco, M.J., principe, L.M., walsh, W.a & Friedman, P.a.Warfarin inhibition of vitamin K2, 3-epoxide reduction in rat microorganisms, biochemistry.22,5655-60 (1983); rost, S., freegin, A., ivaskevicius, V., conzelmann, E. & Hortnagel, K.variants in VKORC1 house warfarin resistance and multiple co-aggregation factor determination type 2.Nature.427,537-541 (2004). Preferably, the following method is used:

mixing the screened micromolecule compound which can inhibit the circulation of the vitamin K in a targeted manner, vitamin K epoxide working solution and VKOR protein working solution, adding the mixture into a fluorescence measurement plate, detecting fluorescence by using a fluorescence detector, setting the wavelength of exciting light to be 245-255nm and the wavelength of emitted light to be 420-440nm, and calculating the increment of a fluorescence value by using an end-point method to represent the activity of VKOR protein; and/or the like, and/or,

and mixing the screened micromolecule compound which can inhibit the circulation of the vitamin K in a targeted manner, the vitamin K working solution and the VKOR protein working solution, adding the mixture into a fluorescence measurement plate, detecting fluorescence by using a fluorescence detector, setting the wavelength of exciting light to be 245-255nm and the wavelength of emitted light to be 420-440nm, and calculating the increment of the fluorescence value by using an end-point method to represent the activity of VKOR protein.

The detection principle of the method is to utilize the KH product ₂ The characteristic of fluorescence can be generated under the excitation light of 250nm, and the KH product can be detected ₂ The activity of VKOR protein can be reflected by the yield of the VKOR protein, and the fluorescence intensity measured by an end-point method can reflect the activity of VKOR protein, or the fluorescence intensity can reflect the activity of VKOR protein detected in real time.

The preparation method of the vitamin K epoxide working solution comprises the following steps: respectively taking a buffer solution 1, a buffer solution 2 and a buffer solution 3, and preparing a vitamin K epoxide working solution with the GSH concentration of 60-160mmol/L and the vitamin K epoxide concentration of 30-80 mu mol/L;

the composition of buffer 1 included: tris-HCl 20-50mmol/L with pH value of 7.0-8.0, naCl 100-150mmol/L, LMNG or GDN 0.5-5g/L, and water as solvent;

the composition of buffer 2 included: 0.4-1mol/L of GSH and water as a solvent; the pH value is 7.0-7.6;

the composition of buffer 3 included: vitamin K epoxide 1-50mmol/L and isopropanol as solvent.

The preparation method of the vitamin K working solution comprises the following steps: respectively taking a buffer solution 1, a buffer solution 2 and a buffer solution 4, and preparing a vitamin K working solution with the GSH concentration of 60-160mmol/L and the vitamin K concentration of 30-80 mu mol/L;

the compositions of the buffer solution 1 and the buffer solution 2 are the same as above;

the composition of buffer 4 included: vitamin K1-100mmol/L and isopropanol as solvent.

The preparation method of the VKOR protein working solution comprises the following steps: respectively taking VKOR protein and buffer solution 1, and preparing VKOR protein working solution with VKOR protein concentration of 1-5 mu mol/L;

the composition of buffer 1 was the same as above.

The buffer solution 2 further comprises an alkaline pH value regulator, such as NaOH, KOH, and the like, and the dosage of the alkaline pH value regulator is based on the regulation of the pH value of the buffer solution 2 to 7.0-7.6.

The buffer solution 1 is added with a surfactant LMNG or GDN to stabilize protein, VKOR protein is transmembrane protein, and can be correctly folded and not denatured only in a proper surfactant.

Further preferably, the buffer 1 has the following composition: tris-HCl 20mmol/L, naCl 100mmol/L, LMNG 1g/L or GDN 0.5g/L at pH 7.5, and water as solvent;

the buffer solution 2 comprises the following components: 0.5mol/L of GSH, a proper amount of alkaline pH value regulator and water as a solvent; the pH value is 7.5;

the buffer solution 3 comprises the following components: 1mmol/L vitamin K epoxide, and isopropanol as solvent;

the buffer solution 4 comprises the following components: vitamin K1mmol/L, and isopropanol as solvent.

The vitamin K epoxide working solution needs to be balanced for more than 1 hour at room temperature after the preparation is finished.

The VKOR protein is a human VKOR protein, and specifically is a purified human VKOR protein. The preparation method of the purified human VKOR protein comprises the following steps: cloning a Gene (Gene ID: 79001) coding human VKOR protein into a pichia pastoris expression vector pPICZ-B (invitrogen), wherein the C terminal of the Gene is sequentially connected with a PreScission protease cleavage site and a GFP-HIS10 label; protein expression and purification were carried out according to the methods in kits (cat # K171001, thermofish Co.) and literature (Ren F, logeman BL, zhang X, liu Y, thiele DJ, yuan P.X-ray constructs of the high-affinity chip transporter Ctr1.Nat Commun.2019,27 (1): 1386.doi 10.1038/s 41467-019-09376-7.). The purified and concentrated human VKOR protein is collected with buffer solution (composed of Tris-HCl 20-50mmol/L, naCl 100-150mmol/L, LMNG or GDN 0.5-5g/L, solvent is water, pH 7.0-8.0) for use.

Further preferably, the preparation method of the vitamin K epoxide working solution comprises the following steps: taking preparation of 1mL of working solution as an example, taking 800. Mu.L of buffer solution 1, 160. Mu.L of buffer solution 2 and 40. Mu.L of buffer solution 3, uniformly mixing, and then balancing at room temperature for more than 1 hour to obtain the vitamin K epoxide working solution with the GSH concentration of 80mmol/L and the vitamin K epoxide concentration of 40. Mu. Mol/L.

Further preferably, the preparation method of the vitamin K working solution comprises the following steps: taking 1mL of prepared working solution as an example, 800. Mu.L of buffer solution 1, 160. Mu.L of buffer solution 2 and 40. Mu.L of buffer solution 4 are uniformly mixed to obtain the vitamin K working solution with the GSH concentration of 80mmol/L and the vitamin K concentration of 40. Mu. Mol/L.

Further preferably, the preparation method of the VKOR protein working solution comprises the following steps: the (purified human) VKOR protein was taken and diluted with buffer 1 to a human VKOR protein concentration of 3. Mu. Mol/L to obtain a VKOR protein working solution.

The increment of the fluorescence value calculated by the end-point method is as follows: detecting fluorescence once every a period of time by using a fluorescence detector, wherein the total detection time is 1-2 hours; activity of VKOR protein = fluorescence value (F) at 1 hour of reaction _T1 ) Background fluorescence value at the beginning of the reaction (F) _T0 ). More preferably: setting the wavelength of exciting light to be 250nm and the wavelength of emitted light to be 430nm; fluorescence was detected every 30 seconds for a total time of 1-2 hours.

In step 3), the construction method of the cell line with two knockouts of VKORC1 and VKORC1L1 genes and simultaneously expressing FIX-Gla-PC fusion gene is the conventional technique in the art (see literature 4). Cell lines with double knockout of VKORC1 and VKORC1L1 genes and simultaneous expression of FIX-Gla-PC fusion genes used in the present invention were complimentary to the authors in reference 4.

The small molecular compound screened by the screening method of the small molecular compound for targeted inhibition of the vitamin K dependent gamma-glutamyl carboxylase and the structural analogue thereof are applied to preparing the inhibitor for targeted inhibition of the vitamin K dependent gamma-glutamyl carboxylase.

The screened micromolecule compound and the structural analogue thereof have the following structural formulas:

(HF13141-H5)、

(structural analog of HF13141-H5 HTS 03341).

The invention has the beneficial effects that:

according to the invention, by constructing a vitamin K circulating small-molecule inhibitor screening system and a small-molecule inhibitor screening system targeting VKOR protein, small-molecule compounds without inhibiting activity of VKOR protein can be screened; and further identifying the screened small molecular compound by using a cell line which is subjected to double knockout of VKORC1 and VKORC1L1 genes and simultaneously expresses a FIX-Gla-PC fusion gene to obtain a small molecular compound HF13141-H5 which can be used for targeted inhibition of vitamin K-dependent gamma-glutamyl carboxylase.

HF13141-H5 was the first small molecule compound identified to specifically inhibit VKGC activity. The IC of HF13141-H5 inhibiting VKGC activity in 293TRex-F9-Met cells was shown ₅₀ 98.5 + -10.33 nM; IC for inhibiting VKGC activity in DKO cells ₅₀ 136.4. + -. 9.56nM. IC for inhibiting VKGC activity in 293TRex-F9-Met cells by structural analogs of HF13141-H5 ₅₀ 916.9 +/-206.8 nM; IC for inhibition of VKGC activity in DKO cells ₅₀ 1154. + -. 301nM. Since the micromolecules for inhibiting the circulation of the vitamin K are lead compounds for developing anticoagulant drugs and rodenticides, the HF13141-H5 and the structural analogues thereof have potential application values for developing anticoagulant drugs and rodenticides.

Drawings

FIG. 1 is a schematic of the vitamin K cycle;

FIG. 2 is the fluorescence values of the 14 kinds of small molecule compounds normalized by KO as a substrate in example 1;

FIG. 3 is the fluorescence values of the 14 kinds of small molecule compounds normalized by using K as a substrate in example 1;

FIG. 4 is the fluorescence values of 7 small molecule compounds normalized by KO as a substrate in example 1;

FIG. 5 is the fluorescence values of 7 kinds of small molecule compounds in example 1 normalized by using K as a substrate;

FIG. 6 is the activity of the reporter gene normalized by the 7 small molecule compounds in example 1;

FIG. 7 shows normalized fluorescence values in test example 1;

FIG. 8 is IC of HF13141-H5 inhibiting VKGC activity in 293TRex-F9-Met cells in test example 2 ₅₀ ；

FIG. 9 is the IC of HF13141-H5 in inhibiting VKGC activity in DKO cells in Experimental example 3 ₅₀ ；

FIG. 10 is the IC of HTS03341 in test example 4 for inhibiting VKGC activity in 293TRex-F9-Met cells ₅₀ ；

FIG. 11 is IC of HTS03341 in Experimental example 5 for inhibiting VKGC activity in DKO cells ₅₀ 。

Detailed Description

The following examples are given only for the purpose of illustrating the present invention in further detail, and are not to be construed as limiting the present invention in any way.

Reagents, materials and instruments:

GSH: glutathione; LMNG: lauryl Maltose neuropentyl Glycol available from Anarrace; GDN: glyco-diosgenin, available from Anarrace; DDM: n-Dodecyl- β -D-Maltopyranoside from Anathrace; CHAPS:3- [ (3-Cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt, 3- [ (3-Cholamidothio) dimethyl lamonoi ] propanesulfonate,3- [ (3-Cholamidothio) dimethyl lamonoi ] -1-propanesulfonate, available from Anarrace; protein expression and purification was performed according to kit, cat # K171001, purchased from Thermofisher; a black 96-well fluorescence measurement plate, which is a Corning (Corning) 3915 total black fluorescence detection plate; fluorescence detector (molecular device, spectramax M5).

The purified human VKOR protein used in the examples and experimental examples was prepared by the following method: cloning a Gene (Gene ID: 79001) coding human VKOR protein into a pichia pastoris expression vector pPICZ-B (invitrogen), wherein the C terminal of the Gene is sequentially connected with a PreScission protease cleavage site and a GFP-HIS10 label; protein expression and purification were carried out according to the method in the kit (cat # K171001, thermofish Co.) and literature (Ren F, logeman BL, zhang X, liu Y, thiele DJ, yuan P.X-ray constructs of the high-affinity chip transporter Ctr1.Nat Commun.2019,27 (1): 1386.Doi 10.1038/s 41467-019-09376-7) for expressing membrane proteins in Pichia pastoris. The purification steps are as follows:

1) Yeast cells expressing human VKOR protein were crushed, dissolved in lysis solution (2% DDM,150mM NaCl,50mM Tris-HCl pH 8.0, 10. Mu.g/mL DNase I,2mM PMSF protease inhibitor, solvent water), and extracted at 4 ℃ for 3 hours (any of 3-4 hours) with stirring; centrifuging the mixture at 30000g at 4 deg.C for 1h, and collecting supernatant;

2) The supernatant was incubated with cobalt column packing (cat 635503, clontech) at 4 ℃ for 3h (3-4 h) with gentle stirring; the cobalt column packing was then collected and washed with 10 column packing volumes of buffer (30mM imidazole,4mM DDM,150mM NaCl, 2mM Tris-HCl pH 8.0, solvent water);

3) Incubating the washed cobalt column filler with PreScission protease at 4 deg.C overnight, removing the C-terminal GFP-HIS10 marker, collecting human VKOR protein, and concentrating with 10kD concentration tube;

4) Passing the concentrated protein through a Superdex 200 molecular sieve with a mobile phase buffer containing 0.1% LMNG,150mM NaCl,20mM Tris-HCl pH 7.5 and water as solvent; collecting the target protein peak, concentrating with 10kD concentration tube to a concentration of more than 30 μmol/L, and freezing at-80 deg.C.

Example 1

The screening method of the small molecule compound targeting to inhibit vitamin K dependent gamma-glutamyl carboxylase in this embodiment comprises the following steps:

Adding the micromolecule compound to be selected into a culture solution of a double-reporter gene cell system containing vitamin K for culturing, and screening out the micromolecule compound which can inhibit the circulation of the vitamin K in a targeted manner, wherein the specific operation is as follows:

a. a293 TRex cell line (invitrogen) (see reference 1) was transfected with a plasmid (shown in SEQ ID NO.5, synthesized by Biotech) simultaneously expressing a FIX-Gla-PC fusion gene and a reporter gene, and a cell line stably expressing both FIX-Gla-PC and Metridia Luciferase was selected using hygromycin and named 293TRex-F9-Met.

b. Small molecule compounds in a Maybridge Hit Finder small molecule compound library (14400 small molecule compounds) are respectively paved in a 96-hole cell culture plate, 150nL of small molecule drugs (the concentration is10 mmol/L) are added in each hole, and the plate is placed in a refrigerator at the temperature of minus 20 ℃ for standby.

c. Laying 293TRex-F9-Met cells in a 96-well plate paved with small molecular compounds, wherein the cell amount of each well is 30000-40000, and the volume is 150 mu L; equivalent to 2-2.5X 10 ⁵ Cell suspension at a concentration of one/mL, the medium was a complete medium containing double antibody (composition: 10% FBS (fetal bovine serum, v/v), penicillin 100IU/mL, streptomycin 100. Mu.g/mL, DMEM (cat # SH30243.01, hyclone)), and the vitamin K concentration was 5. Mu. Mol/L.

d. After 24 hours of culture, the culture supernatants from the 96-well cell culture plates were collected, and the secreted amounts of the reporter gene FIX-gla-PC were measured by ELSIA (see reference 2), and the activity of the beetle Luciferase (Metridia Luciferase) was measured by the Luciferase reporter gene assay (see references 3 and 4), respectively.

e. Calculating the ratio of the secretion amount of FIX-gla-PC to the fluorescence value of the beetle luciferase (the secretion amount of FIX-gla-PC/the fluorescence value of the beetle luciferase is named as R) of each hole, and dividing the ratio of the additive medicines (R drug, abbreviated as Rd) by the ratio of the non-additive medicines (R no flux, abbreviated as Rn) to obtain the ratio (Rd/Rn); and (3) judging the small molecular compound with the ratio Rd/Rn being less than or equal to 0.3 as a positive result, and screening 21 small molecular compounds for targeted vitamin K cycle inhibition, wherein warfarin is used as a positive control, as shown in the following table 1.

TABLE 1 screening results of Maybridge Hit Finder Small molecule Compound library

Numbering	Drug small molecules	Rd/Rn	Number of	Drug small molecules	Rd/Rn
						1	HF13020-E7	0.16	13	HF13110-F4	0.07
2	HF13021-B2	0.09	14	HF13132-G10	0.16
						3	HF13027-G5	0.23	15	HF13136-E7	0.09
4	HF13028-F7	0.19	16	HF13138-A2	0.04
						5	HF13037-D8	0.12	17	HF13141-H5	0.13
6	HF13050-F5	0.13	18	HF13144-B11	0.06
						7	HF13052-C5	0.08	19	HF13148-C7	0.04
8	HF13073-G2	0.18	20	HF13169-G5	0.13
						9	HF13075-C9	0.15	21	HF13178-A2	0.15
10	HF13089-C5	0.22	Positive control	Warfarin	0.08
						11	HF13091-E6	0.27	Untreated group	DMSO		1
12	HF13106-C10	0.14	/	/	/

The structural formula of the 21 small molecule compounds is as follows:

2) Screening small molecule compound without inhibiting VKOR protein activity

Carrying out VKOR protein activity inhibition assay on the screened small molecular compounds for targeted vitamin K cycle inhibition, and screening the small molecular compounds without VKOR protein activity inhibition, wherein the specific operations are as follows:

a. preparation of materials:

respectively preparing a buffer solution 1, a buffer solution 2, a buffer solution 3, a buffer solution 4 and a small molecular compound solution to be selected;

the buffer solution 1 comprises the following components: tris-HCl 20mmol/L, naCl 100mmol/L, LMNG 1g/L and water with pH value of 7.5;

the buffer solution 2 comprises the following components: 0.5mol/L of GSH, a proper amount of NaOH and water as a solvent; the pH value is 7.5;

the buffer solution 3 comprises the following components: 1mmol/L of vitamin K epoxide, and isopropanol as a solvent;

the buffer solution 4 comprises the following components: vitamin K1mmol/L and isopropanol as solvent;

the composition of the small molecule compound solution to be selected is as follows: the concentration of the selected small molecular compound is 1mmol/L, and the solvent is DMSO.

b. Preparing a working solution:

respectively preparing vitamin K epoxide working solution, vitamin K working solution and VKOR protein working solution for later use;

the preparation method of the vitamin K epoxide working solution comprises the following steps: taking preparation of 1mL of working solution as an example, taking 800. Mu.L of buffer solution 1, 160. Mu.L of buffer solution 2 and 40. Mu.L of buffer solution 3, mixing uniformly, and balancing at room temperature for more than 1 hour to obtain a vitamin K epoxide working solution with a GSH concentration of 80mmol/L and a vitamin K epoxide concentration of 40. Mu. Mol/L;

the preparation method of the vitamin K working solution comprises the following steps: taking preparation of 1mL of working solution as an example, taking 800. Mu.L of buffer solution 1, 160. Mu.L of buffer solution 2 and 40. Mu.L of buffer solution 4, and uniformly mixing to obtain a vitamin K working solution with a GSH concentration of 80mmol/L and a vitamin K concentration of 40. Mu. Mol/L;

the preparation method of the VKOR protein working solution comprises the following steps: taking purified human VKOR protein (the concentration is more than 30 mu mol/L), and diluting the purified human VKOR protein with a buffer solution 1 until the concentration of the human VKOR protein is 3 mu mol/L to obtain a VKOR protein working solution.

c. And (3) determining the inhibition effect of the selected small-molecule compound on VKOR protein activity:

determination of inhibitory effect of candidate small molecule compounds on catalytic KO activity of VKOR protein: adding 0.8 mu L of to-be-selected small molecule compound solution into a black 96-hole fluorescence measurement plate, then respectively taking 40 mu L of vitamin K epoxide working solution and 40 mu L of VKOR protein working solution, sequentially adding the vitamin K epoxide working solution and the VKOR protein working solution into the black 96-hole fluorescence measurement plate, uniformly mixing, detecting fluorescence in a 96-hole fluorescence detector (molecular device, spetraMAX M5), setting the wavelength of exciting light to be 250nm, and setting the wavelength of emitted light to be 430nm; detecting fluorescence once every 30 seconds, wherein the total detection time is 1-2 hours, and calculating the increment of the fluorescence value by using an end point method to represent the activity of VKOR protein;

determination of inhibitory effect of candidate small molecule compounds on catalytic K activity of VKOR protein: adding 0.8 mu L of to-be-selected small molecule compound solution into a black 96-hole fluorescence measurement plate, then respectively taking 40 mu L of vitamin K working solution and VKOR protein working solution, sequentially adding the vitamin K working solution and the VKOR protein working solution into the black 96-hole fluorescence measurement plate, uniformly mixing, detecting fluorescence in a 96-hole fluorescence detector (molecular sieve, spetraMAX M5), setting the wavelength of exciting light to be 250nm, and setting the wavelength of emitted light to be 430nm; detecting fluorescence once every 30 seconds, wherein the total detection time is 1-2 hours, and calculating the increment of the fluorescence value by using an end point method to represent the activity of VKOR protein;

activity of VKOR protein = fluorescence value at 1 hour of reaction (F) _T1 ) Background fluorescence value at the beginning of the reaction (F) _T0 )。

Control group: adding 0.8 mu L DMSO into a black 96-hole fluorescence measurement plate, respectively taking 40 mu L vitamin K epoxide working solution (or vitamin K working solution) and VKOR protein working solution, sequentially adding into the black 96-hole fluorescence measurement plate, and uniformly mixing, wherein the rest are the same as above.

Warfarin control group: 0.8 mu L warfarin solution (warfarin 0.4mmol/L, solvent is DMSO) is added into a black 96-hole fluorescence measurement plate, then, 40 mu L of each of vitamin K epoxide working solution (or vitamin K working solution) and VKOR protein working solution is respectively taken, and the mixture is sequentially added into the black 96-hole fluorescence measurement plate and uniformly mixed, and the rest is the same as the above.

d. Determination of inhibitory Effect

The activity of the control group was noted as CF _A Warfaring pairReference Activity was recorded as WF _A Small molecule Compound treatment group Activity is denoted as DF _A . Thereafter, the fluorescence value was compared with the CF of the control group _A Performing homogenization treatment, wherein the control group is1 and represents the state of no inhibition of VKOR protein activity; warfarin treatment group is WF _A /CF _A (normalized fluorescence value), indicating the inhibitory effect of warfarin on VKOR protein activity; activity of small molecule Compound treated groups divided by Activity of control groups, i.e. DF _A /CF _A (normalized fluorescence value) indicates the inhibitory effect of small molecule compounds on VKOR protein activity. If DF is _A /CF _A The numerical value is less than or equal to 1/2, which shows that the small molecular compound has better inhibition effect on VKOR protein activity; if DF is _A /CF _A ≤WF _A /CF _A It means that the inhibitory effect of the small molecule compound is equal to or better than that of warfarin; if 1/2DF _A /CF _A ＞WF _A /CF _A Indicating that the small molecule compounds are less effective than warfarin in inhibition; if 1> DF _A /CF _A The value is more than 1/2, which indicates that the inhibition effect of the small molecular compound is poor; if DF is _A /CF _A The value is1 or more, indicating no inhibitory effect.

The screening results are shown in table 2 below, and it can be seen from table 2 that 14 small molecules with good VKOR inhibitory activity and 7 small molecules with poor or no VKOR inhibitory activity are screened out.

TABLE 2 VKOR protein activity inhibition results of 21 small molecule compounds

Analysis of inhibitory effect of 14 small molecule compounds on VKOR protein activity (as shown in fig. 2-3): the 14 small molecule compounds are respectively: HF13020-E7, HF13021-B2, HF13028-F7, HF13052-C5, HF13073-G2, HF13089-C5, HF13091-E6, HF13106-C10, HF13110-F4, HF13136-E7, HF13138-A2, HF13169-G5, HF13144-B11, HF13148-C7. Vitamin K epoxide (KO) is taken as a substrate, and 14 small molecular compounds can be detected to well inhibit VKOR protein activity. Wherein the 7 small molecular compounds have better inhibition effect than warfarin in vitro activity assay, and are respectively HF13073-G2, HF13106-C10, HF13110-F4, HF13136-E7, HF13138-A2, HF13144-B11 and HF13148-C7. Among the 14 small molecular compounds, vitamin K (K) is used as a substrate, and the inhibition effect of the vitamin K (K) on the activity of VKOR protein catalysis K is detected, and 5 small molecular compounds are found to have poor inhibition effects, namely HF13052-C5, HF13073-G2, HF13089-C5, HF13091-E6 and HF13169-G5; the inhibition effect of the 3 small molecular compounds is not as good as that of warfarin, namely HF13028-F7, HF13106-C10 and HF13144-B11; the inhibition effect of the 6 small molecular compounds is equal to or better than that of warfarin, namely HF13020-E7, HF13021-B2, HF13110-F4, HF13136-E7, HF13138-A2 and HF13148-C7.

Analysis of inhibitory effects of 7 small molecule compounds on VKOR protein activity (as shown in fig. 4-5): the 7 small molecule compounds are respectively: HF13027-G5, HF13037-D8, HF13050-F5, HF13075-C9, HF13132-G10, HF13141-H5, HF13178-A2. Vitamin K epoxide (KO) or vitamin K is taken as a substrate respectively, and the inhibitory effect of the vitamin K epoxide (KO) or the vitamin K on the catalytic activity of VKOR protein is detected, so that the vitamin K epoxide (KO) or the vitamin K is found to have poor or no inhibitory effect on the VKOR protein.

3) Screening of small molecule compounds for targeted inhibition of vitamin K dependent gamma-glutamyl carboxylase

The screened small molecule compound is identified by using a cell line which is subjected to double knockout of VKORC1 and VKORC1L1 genes and simultaneously expresses a FIX-Gla-PC fusion gene (see literature 4), so as to obtain the small molecule compound for targeted inhibition of vitamin K-dependent gamma-glutamyl carboxylase, which comprises the following steps:

acquisition of a cell line with double knockout of VKORC1 and VKORC1L1 genes (DKO cell line) simultaneously expressing FIX-Gla-PC fusion gene

DKO cell lines were given as a gift by the authors in document 4.

b. In a 24-well cell culture plate, 0.8. Mu.L of small molecule drug (10 mmol/L) was added to each well, and 3 wells were plated for each small molecule, wherein DMSO and warfarin groups were used as controls.

c. Laying DKO cell line cells in 24-well plates paved with small molecular compounds, wherein the cell amount of each well is 160000-200000 cells, and the volume is 800 mu L; equivalent to 2-2.5X 10 ⁵ Cell suspension at a concentration of one/mL, the medium was a complete medium containing double antibody (composition: 10% FBS (fetal bovine serum, v/v), penicillin 100IU/mL, streptomycin 100. Mu.g/mL, DMEM (cat # SH30243.01, hyclone)), and the vitamin K concentration was 10. Mu. Mol/L.

d. After 48 hours of culture, the culture supernatants from the 24-well plates were collected, and the secreted amounts of the reporter gene FIX-gla-PC were measured by ELSIA, respectively.

e. The effect of small molecule compounds on reporter activity in DKO cells was calculated: the reporter gene activity of the drug-added group is divided by the reporter gene activity of the drug-not-added group (DMSO-treated group), and the obtained ratio is the normalized reporter gene activity. The reporter gene activity homogenized in the DMSO treatment group is1, and the small molecule compound with the reporter gene activity homogenized in the drug-added treatment group being less than or equal to 0.3 is judged as a positive result. The identification result is shown in FIG. 6, and 1 small molecule compound HF13141-H5 is co-screened, and is a small molecule for inhibiting VKGC activity.

And (4) analyzing results: as can be seen from the results of the DKO cell reporter gene activity assay, the small molecule compound HF13141-H5 specifically inhibits the VKGC enzyme activity, while other small molecules do not inhibit the VKGC enzyme activity.

Example 2

In the embodiment, the small molecule compound HF13141-H5 and the structural analogue HTS03341 screened in the embodiment 1 are used for preparing the inhibitor of the targeted vitamin K-dependent gamma-glutamyl carboxylase, and the structural formula of the small molecule compound HF13141-H5 and the structural analogue HTS03341 is as follows:

(HF13141-H5)、

(HTS03341)。

test example 1

VKOR in vitro activity assay: the inhibition effect of the small molecule compound HF13141-H5 on VKOR activity is determined by the following method:

a. preparation of materials:

respectively preparing a buffer solution 1, a buffer solution 2, a buffer solution 3, a buffer solution 4, a warfarin solution and an HF13141-H5 solution;

the buffer solution 1 comprises the following components: tris-HCl 20mmol/L, naCl 100mmol/L, LMNG 1g/L, pH 7.5, solvent water;

the buffer solution 4 comprises the following components: vitamin K1mmol/L, and isopropanol as solvent;

the warfarin solution comprises the following components: warfarin 0.4mmol/L, solvent DMSO;

the composition of the HF13141-H5 solution is as follows: HF13141-H5 is 0.4mmol/L, and the solvent is DMSO.

b. Preparing a working solution:

respectively preparing a vitamin K epoxide working solution, a vitamin K working solution and a VKOR protein working solution;

the preparation method of the vitamin K epoxide working solution comprises the following steps: taking preparation of 1mL of working solution as an example, taking 800 mu L of buffer solution 1, 160 mu L of buffer solution 2 and 40 mu L of buffer solution 3, uniformly mixing, and balancing at room temperature for more than 1 hour to obtain a vitamin K epoxide working solution with GSH concentration of 80mmol/L and vitamin K epoxide concentration of 40 mu mol/L;

c. Determination of inhibition effect of small molecule compound HF13141-H5 on VKOR protein activity:

determination of inhibition effect of small molecule compound HF13141-H5 on VKOR protein catalysis KO activity: adding 0.8 mu L of HF13141-H5 solution into a black 96-hole fluorescence measurement plate, then respectively taking 40 mu L of vitamin K epoxide working solution and 40 mu L of VKOR protein working solution, sequentially adding the vitamin K epoxide working solution and the VKOR protein working solution into the black 96-hole fluorescence measurement plate, uniformly mixing, detecting fluorescence in a 96-hole fluorescence detector (molecular device, spetramax M5), setting the wavelength of exciting light to be 250nm and the wavelength of emitted light to be 430nm; detecting fluorescence once every 30 seconds, wherein the total detection time is 1-2 hours, and calculating the increment of the fluorescence value by using an end-point method to represent the activity of VKOR protein;

determination of inhibition of small molecule compound HF13141-H5 on VKOR protein catalytic K activity: adding 0.8 mu L of HF13141-H5 solution into a black 96-hole fluorescence measurement plate, then respectively taking 40 mu L of vitamin K working solution and VKOR protein working solution, sequentially adding the vitamin K working solution and the VKOR protein working solution into the black 96-hole fluorescence measurement plate, uniformly mixing, detecting fluorescence in a 96-hole fluorescence detector (molecular device, spetramax M5), setting the wavelength of exciting light to be 250nm and the wavelength of emitted light to be 430nm; detecting fluorescence once every 30 seconds, wherein the total detection time is 1-2 hours, and calculating the increment of the fluorescence value by using an end-point method to represent the activity of VKOR protein;

activity of VKOR protein = fluorescence value (F) at 1 hour of reaction _T1 ) Background fluorescence value at the beginning of the reaction (F) _T0 )。

Warfarin control group: 0.8 mu L warfarin solution is added into a black 96-hole fluorescence measurement plate, then, 40 mu L of each of the vitamin K epoxide working solution (or the vitamin K working solution) and the VKOR protein working solution is respectively taken, and then, the mixture is sequentially added into the black 96-hole fluorescence measurement plate and uniformly mixed, and the rest is the same as the above.

Control groupThe activity of (A) is noted as CF _A Warfaring control activity was designated as WF _A The activity of the small molecule compound treatment group is marked as DF _A . Thereafter, the fluorescence value was compared with the CF of the control group _A Performing homogenization treatment, wherein the control group is1 and represents the state of non-inhibition of VKOR protein activity; warfarin treatment group is WF _A /CF _A (normalized fluorescence value), indicating the inhibitory effect of warfarin on VKOR protein activity; the activity of the HF13141-H5 treated group was divided by the activity of the control group, i.e. DF _A /CF _A (normalized fluorescence value) indicating the inhibitory effect of HF13141-H5 on VKOR protein activity; the test results are shown in FIG. 7.

As can be seen from FIG. 7, the small molecule compound HF13141-H5 did not inhibit VKOR activity.

Test example 2

Determination of IC of small molecule HF13141-H5 for VKGC inhibition in 293TRex-F9-Met cells ₅₀ The method comprises the following steps:

1) Preparing culture medium with 12 concentration gradients of small molecule compound HF13141-H5, wherein the concentration of vitamin K is 5 mu mol/L, and the culture medium is DMEM complete culture medium; HF13141-H5 concentrations of 4. Mu.M, 2. Mu.M, 1. Mu.M, 0.5. Mu.M, 0.25. Mu.M, 0.125. Mu.M, 0.0625. Mu.M, 0.03125. Mu.M, 0.0156. Mu.M, 0.0078. Mu.M, 0.0039. Mu.M, 0. Mu.M, respectively; this was followed by addition to 24 well cell culture plates at 400. Mu.L per well.

2) A cell suspension of 93TRex-F9-Met cells was prepared, and the cells were cultured in complete DMEM medium (composition: 10% of FBS (fetal bovine serum, v/v), penicillin 100IU/mL, streptomycin 100. Mu.g/mL, DMEM (cat # SH30243.01, hyclone)) to prepare a cell suspension at a cell concentration of 2-2.5X 10 ⁵ cells/mL, 400. Mu.L of cell suspension was added to the above 24-well cell culture plate containing HF13141-H5.

3) After 24 hours of culture, the culture supernatant was collected, and the secreted amount of the reporter gene FIX-gla-PC was measured by ELSIA (see reference 2), and the activity of the beetle Luciferase (Metridia Luciferase) was measured by the Luciferase reporter gene assay (see references 3 and 4), respectively.

4) Calculating IC of Small molecule Compounds ₅₀ : firstly, the secretion amount of FIX-gla-PC under different concentrations is calculatedThe ratio of the fluorescence value of the beetle luciferase (the secretion amount of FIX-gla-PC/the fluorescence value of the beetle luciferase, named as R) is obtained by homogenizing the R value of the drug treatment groups with different concentrations by the R value of the non-drug-added group, and then calculating the IC of the small molecular compound HF13141-H5 for inhibiting the VKGC activity by the graphpad prism 5 ₅₀ The results are shown in FIG. 8.

Warfarin treatment was used as a control group, and warfarin concentrations were 1. Mu.M, 0.5. Mu.M, 0.25. Mu.M, 0.125. Mu.M, 0.0625. Mu.M, 0.03125. Mu.M, 0.0156. Mu.M, 0.0078. Mu.M, 0.0039. Mu.M, 0.00196. Mu.M, 0.00098. Mu.M, and 0. Mu.M, respectively; the measurement method was the same as above.

Test results show that HF13141-H5 inhibits IC of VKGC activity in 293TRex-F9-Met cells ₅₀ 98.5 + -10.33 nM, while the control group warfarin (warfarin) inhibits the IC of vitamin K cycle ₅₀ Was 86.1. + -. 7.28nM.

Test example 3

Determination of IC of Small molecules HF13141-H5 for VKGC Activity inhibition in DKO cells ₅₀ The method comprises the following steps:

1) Preparing culture medium with 12 concentration gradients of small molecule compound HF13141-H5, wherein the concentration of vitamin K is10 mu mol/L, and the culture medium is DMEM complete culture medium; HF13141-H5 concentrations of 4. Mu.M, 2. Mu.M, 1. Mu.M, 0.5. Mu.M, 0.25. Mu.M, 0.125. Mu.M, 0.0625. Mu.M, 0.03125. Mu.M, 0.0156. Mu.M, 0.0078. Mu.M, 0.0039. Mu.M, 0. Mu.M, respectively; this was followed by addition to 24 well cell culture plates at 400. Mu.L per well.

2) A cell suspension of DKO cells was prepared, and the cells were cultured in complete DMEM medium (composition: 10% of FBS (fetal bovine serum, v/v), penicillin 100IU/mL, streptomycin 100. Mu.g/mL, DMEM (cat # SH30243.01, hyclone)) to prepare a cell suspension at a cell concentration of 2-2.5X 10 ⁵ cells/mL, 400. Mu.L of cell suspension was added to the above 24-well cell culture plate containing HF13141-H5.

3) After 48 hours of culture, the culture supernatants from the 24-well plates were collected, and the secreted amounts of the reporter gene FIX-gla-PC were measured by ELSIA, respectively.

4) Calculating IC of small molecule compound HF13141-H5 ₅₀ : reporting of drug treatment groups at different concentrations with reporter Gene Activity without drug addition groupHomogenizing gene activity, and calculating IC of VKGC activity inhibition by small molecule compound HF13141-H5 using Graphpad prism 5 ₅₀ The results are shown in FIG. 9.

Warfarin and DMSO treatments were used as controls, and warfarin concentrations were 4. Mu.M, 2. Mu.M, 1. Mu.M, 0.5. Mu.M, 0.25. Mu.M, 0.125. Mu.M, 0.0625. Mu.M, 0.03125. Mu.M, 0.0156. Mu.M, 0.0078. Mu.M, 0.0039. Mu.M, 0. Mu.M, respectively; the measurement method is the same as above.

Test results show that HF13141-H5 inhibits IC of VKGC activity in DKO cells ₅₀ 136.4 ± 9.56nM; warfarin and DMSO treated controls, which had no inhibitory effect on VKGC enzyme activity.

Test example 4

The structural analog of HF13141-H5, HTS03341, was purchased from molport, compound number (Compound number): molPort-001-807-211, company address https:// www. MolPort. Com/shop/find-chemicals.

Determination of IC of Small molecule HTS03341 for inhibiting VKGC Activity in 293TRex-F9-Met cells ₅₀ The method comprises the following steps:

1) Preparing culture medium with 12 concentration gradients of small molecule compound HTS03341, wherein the concentration of vitamin K is 5 mu mol/L, and the culture medium is DMEM complete culture medium; the concentrations of HTS03341 were 10. Mu.M, 5. Mu.M, 2.5. Mu.M, 1.25. Mu.M, 0.625. Mu.M, 0.3125. Mu.M, 0.156. Mu.M, 0.078. Mu.M, 0.039. Mu.M, 0.0195. Mu.M, 0.0098. Mu.M, 0. Mu.M, respectively; this was followed by addition to a 24 well cell culture plate at 400. Mu.L per well.

2) A cell suspension of 93TRex-F9-Met cells was prepared, and the cells were cultured in complete DMEM medium (composition: 10% of FBS (fetal bovine serum, v/v), penicillin 100IU/mL, streptomycin 100. Mu.g/mL, DMEM (cat # SH30243.01, hyclone)) to prepare a cell suspension at a cell concentration of 2-2.5X 10 ⁵ cells/mL, 400 μ L of cell suspension was added to the above 24-well cell culture plate containing HTS 03341.

4) Calculating IC of Small molecule Compounds ₅₀ : firstly, calculating the ratio of the secretion of FIX-gla-PC to the fluorescence value of the beetle luciferase (the secretion of FIX-gla-PC/the fluorescence value of the beetle luciferase, named as R) under different concentrations, carrying out homogenization treatment on the R values of the drug treatment groups with different concentrations by using the R values of the non-drug-added groups, and then calculating the IC of the small-molecular compound HTS03341 for inhibiting the VKGC activity by using graphpad prism 5 ₅₀ The results are shown in FIG. 10.

The results of the experiments show that HTS03341 inhibits the IC of VKGC activity in 293TRex-F9-Met cells ₅₀ It was 916.9. + -. 206.8nM.

Test example 5

Determination of IC for Small molecule HTS03341 to inhibit VKGC Activity in DKO cells ₅₀ The method comprises the following steps:

1) Preparing culture medium with 12 concentration gradients of small molecule compound HTS03341, wherein the concentration of vitamin K is10 mu mol/L, and the culture medium is DMEM complete culture medium; HTS03341 at a concentration of 10. Mu.M, 5. Mu.M, 2.5. Mu.M, 1.25. Mu.M, 0.625. Mu.M, 0.3125. Mu.M, 0.156. Mu.M, 0.078. Mu.M, 0.039. Mu.M, 0.0195. Mu.M, 0.0098. Mu.M, 0. Mu.M, respectively; this was followed by addition to a 24 well cell culture plate at 400. Mu.L per well.

2) A cell suspension of DKO cells was prepared, and the cells were cultured in complete DMEM medium (composition: 10% of FBS (fetal bovine serum, v/v), penicillin 100IU/mL, streptomycin 100. Mu.g/mL, DMEM (cat # SH30243.01, hyclone)) to prepare a cell suspension at a cell concentration of 2-2.5X 10 ⁵ cells/mL, 400 μ L of cell suspension was added to the above 24-well cell culture plate containing HTS 03341.

4) IC calculation of Small molecule Compound HTS03341 ₅₀ : the reporter gene activity of the drug treatment groups with different concentrations is homogenized by the reporter gene activity of the drug-free group, and then the IC of inhibiting VKGC activity by the small molecule compound HTS03341 is calculated by Graphpad prism 5 ₅₀ The results are shown in FIG. 11.

The results of the experiments show that HTS03341 is in DKO cellsIC for inhibiting VKGC activity ₅₀ 1154. + -.301 nM.

Reference documents:

document 1: wanrooij S, goffart S,

JLO,Yasukawa T,Spelbrink JN.Expression of catalytic mutants of the mtDNAhelicase Twinkle and polymerase POLG causes distinct replication stalling phenotypes.Nucleic Acids Res 2007；35:3238-51.；

document 2: tie J-K, jin D-Y, stright DL, stafford DW.functional study of the vitamin K cycle in a mammarian cell.blood 2011; 117;

document 3: shen G, cui W, zhang H, zhou F, huang W, liu Q, yang Y, li S, bowman GR, sadler JE, gross ML, li W, guomin S, weidong C, hao Z, fengbo Z, weih, qian L, yihu Y, bowman GR, et al, surface prediction blocks coargulation by mapping human vitamin K epoxide reduction in an intermediate stage product yield. 24;

document 4: tie JK, jin DY, tie K, stable DW. Evaluation of warp resistance using transformation activators-like effector genes-modified vitamin K epoxide reaction knottout HEK293 cells J Thromb Haemost 2013;11:1556-64..

Sequence listing

SEQUENCE LISTING

<110> university of Henan science and technology

<120> screening method and application of small molecular compound for targeted inhibition of vitamin K-dependent gamma-glutamyl carboxylase

<170> SIPOSequenceListing 1.0

<160> 5

<211> 1392

<212> DNA

<213> recombination of human F9 and PROC genes

<221> FIX-Gla-PC fusion gene

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cgaaaacgtt ataattcagg taaattggaa gagtttgttc aagggaacct tgagagagaa 180

tgtatggaag aaaagtgtag ttttgaagaa gcacgagaag tttttgaaaa cacggaaaga 240

acaactgaat tttggaagca gtatgttgac ggtgaccagt gcttggtctt gcccttggag 300

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ggggcagtgc tcatccaccc ctcctgggtg ctgacagcgg cccactgcat ggatgagtcc 780

aagaagctcc ttgtcaggct tggagagtat gacctgcggc gctgggagaa gtgggagctg 840

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gagggcgaca gtggggggcc catggtcgcc tccttccacg gcacctggtt cctggtgggc 1260

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tgggcacctt aa 1392

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<213> recombination of human F9 and PROC genes

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MAWQLTSLLL FVATWGISGT PAPLDSVFSS SERAHQVLRI RKRYNSGKLE EFVQGNLERE 60

CMEEKCSFEE AREVFENTER TTEFWKQYVD GDQCLVLPLE HPCASLCCGH GTCIDGIGSF 120

SCDCRSGWEG RFCQREVSFL NCSLDNGGCT HYCLEEVGWR RCSCAPGYKL GDDLLQCHPA 180

VKFPCGRPWK RMEKKRSHLK RDTEDQEDQV DPRLIDGKMT RRGDSPWQVV LLDSKKKLAC 240

GAVLIHPSWV LTAAHCMDES KKLLVRLGEY DLRRWEKWEL DLDIKEVFVH PNYSKSTTDN 300

DIALLHLAQP ATLSQTIVPI CLPDSGLAER ELNQAGQETL VTGWGYHSSR EKEAKRNRTF 360

VLNFIKIPVV PHNECSEVMS NMVSENMLCA GILGDRQDAC EGDSGGPMVA SFHGTWFLVG 420

LVSWGEGCGL LHNYGVYTKV SRYLDWIHGH IRDKEAPQKS WAP 463

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gaaaccgacc tgttcaccat ctgggagacc atggaagtga tgatcaaggc cgacatcgcc 180

gacaccgacc gggccagcaa cttcgtggcc accgagaccg acgccaaccg gggcaagatg 240

cccggcaaga agctgcccct ggccgtcatc atggaaatgg aagccaacgc cttcaaggcc 300

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gtgtacatcc ccggcaggtg ccacgactac ggcggcgaca agaaaaccgg ccaggccggc 420

atcgtgggcg ccatcgtgga catccccgag atcagcggct tcaaagaaat ggcccccatg 480

gaacagttca tcgcccaggt ggacagatgc gccagctgca ccaccggctg cctgaagggc 540

ctggccaacg tgaagtgcag cgagctgctg aagaagtggc tgcccgaccg ctgcgccagc 600

ttcgccgaca agatccagaa agaggtgcac aacatcaagg gcatggccgg cgacaggtga 660

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<213> beetles

<221> protein encoded by beetle luciferase reporter gene

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MDIKVVFTLV FSALVQAKST EFDPNIDIVG LEGKFGITNL ETDLFTIWET MEVMIKADIA 60

DTDRASNFVA TETDANRGKM PGKKLPLAVI MEMEANAFKA GCTRGCLICL SKIKCTAKMK 120

VYIPGRCHDY GGDKKTGQAG IVGAIVDIPE ISGFKEMAPM EQFIAQVDRC ASCTTGCLKG 180

LANVKCSELL KKWLPDRCAS FADKIQKEVH NIKGMAGDR 219

<211> 9452

<212> DNA

<213> Artificial sequence

<221> plasmid simultaneously expressing FIX-Gla-PC fusion gene and reporter gene

<400> 5

ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca 60

aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg 120

cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag 180

taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt 240

acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg 300

acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat 360

ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct 420

attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg 480

gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg 540

ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc 600

caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa 660

tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 720

tatataagca gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt 780

aatacgactc actataggga gacccaagct tgcattcctg caggtcgacg ccgccaccat 840

ggacatcaag gtggtgttca ccctggtgtt cagcgccctg gtgcaggcca agagcaccga 900

gttcgacccc aacatcgaca tcgtgggcct ggaaggcaag ttcggcatca ccaacctgga 960

aaccgacctg ttcaccatct gggagaccat ggaagtgatg atcaaggccg acatcgccga 1020

caccgaccgg gccagcaact tcgtggccac cgagaccgac gccaaccggg gcaagatgcc 1080

cggcaagaag ctgcccctgg ccgtcatcat ggaaatggaa gccaacgcct tcaaggccgg 1140

ctgcacccgg ggctgcctga tctgcctgag caagatcaag tgcaccgcca agatgaaggt 1200

gtacatcccc ggcaggtgcc acgactacgg cggcgacaag aaaaccggcc aggccggcat 1260

cgtgggcgcc atcgtggaca tccccgagat cagcggcttc aaagaaatgg cccccatgga 1320

acagttcatc gcccaggtgg acagatgcgc cagctgcacc accggctgcc tgaagggcct 1380

ggccaacgtg aagtgcagcg agctgctgaa gaagtggctg cccgaccgct gcgccagctt 1440

cgccgacaag atccagaaag aggtgcacaa catcaagggc atggccggcg acaggtgata 1500

atctagagga tccgaacaaa aactcatctc agaagaggat ctgaatatgc ataccggtca 1560

tcatcaccat caccattgag tttgatcccc gggaattcag acatgataag atacattgat 1620

gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt 1680

gatgctattg ctttatttgt aaccattata agctgcaata aacaagttgg ggtgggcgaa 1740

gaactccagc atgagatccc cgcgctggag gatcatccag ccggcgtccc ggaaaacgat 1800

tccgaagccc aacctttcat agaaggcggc ggtggaatcg aaatctcgta gcacgtgtca 1860

gtcctgctcc tcggccacga agtgcacgca gttgccggcc gggtcgcgca gggcgaactc 1920

ccgcccccac ggctgctcgc cgatctcggt catggccggc ccggaggcgt cccggaagtt 1980

cgtggacacg acctccgacc actcggcgta cagctcgtcc aggccgcgca cccacaccca 2040

ggccagggtg ttgtccggca ccacctggtc ctggaccgcg ctgatgaaca gggtcacgtc 2100

gtcccggacc acaccggcga agtcgtcctc cacgaagtcc cgggagaacc cgagccggtc 2160

ggtccagaac tcgaccgctc cggcgacgtc gcgcgcggtg agcaccggaa cggcactggt 2220

caacttggcc atggtttagt tcctcacctt gtcgtattat actatgccga tatactatgc 2280

cgatgattaa ttgtcaacac gtgctgatca gatccgaaaa tggatataca agctcccggg 2340

agctttttgc aaaagcctag gcctccaaaa aagcctcctc actacttctg gaatagctca 2400

gaggcagagg cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga 2460

gaatgggcgg aactgggcgg agttaggggc gggatgggcg gagttagggg cgggactatg 2520

gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac 2580

tttccacacc tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg 2640

gagcctgggg actttccaca ccctcgtcga gctagcttcg tgaggctccg gtgcccgtca 2700

gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg 2760

aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct 2820

ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt 2880

tctttttcgc aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg 2940

gcctggcctc tttacgggtt atggcccttg cgtgccttga attacttcca cctggctcca 3000

gtacgtgatt cttgatcccg agctggagcc aggggcgggc cttgcgcttt aggagcccct 3060

tcgcctcgtg cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg 3120

tggcaccttc gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga 3180

tgacctgctg cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaggatctg 3240

cacactggta tttcggtttt tgggcccgcg gccggcgacg gggcccgtgc gtcccagcgc 3300

acatgttcgg cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct 3360

caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg 3420

gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc 3480

cctgctccag ggggctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca 3540

cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag 3600

taccgggcgc cgtccaggca cctcgattag ttctggagct tttggagtac gtcgtcttta 3660

ggttgggggg aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa 3720

gttaggccag cttggcactt gatgtaattc tcgttggaat ttgccctttt tgagtttgga 3780

tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg 3840

tcgtgaacac gtggtcgcgg ccgcttcgaa ggtaccgagc tcggatccgc cgccaccatg 3900

gcatggcagc tcacaagcct cctcctgttc gtggccacct ggggaatttc cggcacacca 3960

gctcctcttg actcagtgtt ctccagcagc gagcgtgccc accaggtgct gcggattcga 4020

aaacgttata attcaggtaa attggaagag tttgttcaag ggaaccttga gagagaatgt 4080

atggaagaaa agtgtagttt tgaagaagca cgagaagttt ttgaaaacac ggaaagaaca 4140

actgaatttt ggaagcagta tgttgacggt gaccagtgct tggtcttgcc cttggagcac 4200

ccgtgcgcca gcctgtgctg cgggcacggc acgtgcatcg acggcatcgg cagcttcagc 4260

tgcgactgcc gcagcggctg ggagggccgc ttctgccagc gcgaggtgag cttcctcaat 4320

tgctctctgg acaacggcgg ctgcacgcat tactgcctag aggaggtggg ctggcggcgc 4380

tgtagctgtg cgcctggcta caagctgggg gacgacctcc tgcagtgtca ccccgcagtg 4440

aagttccctt gtgggaggcc ctggaagcgg atggagaaga agcgcagtca cctgaaacga 4500

gacacagaag accaagaaga ccaagtagat ccgcggctca ttgatgggaa gatgaccagg 4560

cggggagaca gcccctggca ggtggtcctg ctggactcaa agaagaagct ggcctgcggg 4620

gcagtgctca tccacccctc ctgggtgctg acagcggccc actgcatgga tgagtccaag 4680

aagctccttg tcaggcttgg agagtatgac ctgcggcgct gggagaagtg ggagctggac 4740

ctggacatca aggaggtctt cgtccacccc aactacagca agagcaccac cgacaatgac 4800

atcgcactgc tgcacctggc ccagcccgcc accctctcgc agaccatagt gcccatctgc 4860

ctcccggaca gcggccttgc agagcgcgag ctcaatcagg ccggccagga gaccctcgtg 4920

acgggctggg gctaccacag cagccgagag aaggaggcca agagaaaccg caccttcgtc 4980

ctcaacttca tcaagattcc cgtggtcccg cacaatgagt gcagcgaggt catgagcaac 5040

atggtgtctg agaacatgct gtgtgcgggc atcctcgggg accggcagga tgcctgcgag 5100

ggcgacagtg gggggcccat ggtcgcctcc ttccacggca cctggttcct ggtgggcctg 5160

gtgagctggg gtgagggctg tgggctcctt cacaactacg gcgtttacac caaagtcagc 5220

cgctacctcg actggatcca tgggcacatc agagacaagg aagcccccca gaagagctgg 5280

gcaccttaat ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt 5340

tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact 5400

cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat 5460

tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc 5520

aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc 5580

tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 5640

acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 5700

ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 5760

ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat 5820

ggttcacgta cctagaagtt cctattccga agttcctatt ctctagaaag tataggaact 5880

tccttggcca aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag 5940

ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc 6000

ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac 6060

aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt 6120

gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc 6180

acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc 6240

atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg 6300

caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat 6360

gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc 6420

gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat 6480

ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc 6540

gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg 6600

ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga 6660

tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg 6720

gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga 6780

tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc 6840

gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg 6900

gcaaaggaat agcacgtact acgagatttc gattccaccg ccgccttcta tgaaaggttg 6960

ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg 7020

ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc 7080

aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg 7140

tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg 7200

gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 7260

aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc 7320

acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 7380

cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 7440

tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 7500

tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 7560

gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 7620

aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 7680

ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 7740

gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 7800

ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 7860

ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 7920

cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 7980

attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 8040

ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 8100

aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 8160

gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 8220

tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 8280

ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 8340

taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 8400

atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 8460

actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 8520

cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 8580

agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 8640

gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 8700

gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 8760

gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 8820

gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 8880

cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 8940

ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 9000

accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 9060

aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 9120

aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 9180

caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 9240

ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 9300

gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 9360

cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct 9420

gctctgatgc cgcatagtta agccagtatc tg 9452

Claims

1. A method for screening a small molecule compound which can target and inhibit vitamin K dependent gamma-glutamyl carboxylase, which is characterized in that: the method comprises the following steps:

2) Screening small molecule compound without inhibiting VKOR protein activity

Identifying the screened small molecular compound by using a cell line which is subjected to double knockout of VKORC1 and VKORC1L1 genes and simultaneously expresses a FIX-Gla-PC fusion gene to obtain the small molecular compound for targeted inhibition of vitamin K dependent gamma-glutamyl carboxylase;

in step 2), the method for determining the inhibition of VKOR protein activity is as follows:

mixing the screened small molecular compound for targeted inhibition of vitamin K circulation with vitamin K epoxide working solution and VKOR protein working solution, adding into a fluorescence measurement plate, detecting fluorescence by using a fluorescence detector, setting the wavelength of exciting light to be 245-255nm and the wavelength of emitted light to be 420-440nm, and calculating the increment of the fluorescence value by using an end-point method to represent the activity of VKOR protein; and/or the like, and/or,

mixing the screened small molecular compound for targeted inhibition of vitamin K circulation with vitamin K working solution and VKOR protein working solution, adding into a fluorescence measurement plate, detecting fluorescence by using a fluorescence detector, setting the wavelength of exciting light to be 245-255nm and the wavelength of emitted light to be 420-440nm, and calculating the increment of the fluorescence value by using an end-point method to represent the activity of VKOR protein;

the preparation method of the VKOR protein working solution comprises the following steps: respectively taking VKOR protein and buffer solution 1, and preparing a VKOR protein working solution with the VKOR protein concentration of 1-5 mu mol/L;

the composition of buffer 3 included: 1-50mmol/L of vitamin K epoxide, and isopropanol as a solvent;

2. The screening method of small molecule compounds targeted to inhibit vitamin K-dependent γ -glutamyl carboxylase according to claim 1, characterized in that: in the step 1), the host cell of the double-reporter gene cell system is a human embryonic kidney cell 293Trex.

3. The screening method of small molecule compounds targeted to inhibit vitamin K-dependent γ -glutamyl carboxylase according to claim 1, characterized in that: in the step 1), the reporter gene is a beetle luciferase reporter gene, the nucleotide sequence of the beetle luciferase reporter gene is shown as SEQ ID NO.3, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 4.

4. The screening method of small molecule compounds targeted to inhibit vitamin K-dependent γ -glutamyl carboxylase according to claim 1, characterized in that: in the step 1), the nucleotide sequence of the plasmid for simultaneously expressing the FIX-Gla-PC fusion gene and the reporter gene is shown as SEQ ID NO. 5.

5. The screening method of small molecule compounds targeted to inhibit vitamin K-dependent γ -glutamyl carboxylase according to claim 1, characterized in that: in the step 1), the concentration of the vitamin K in the culture solution of the double-reporter gene cell system is 1-20 mu mol/L.

6. The screening method of small molecule compounds targeted to inhibit vitamin K-dependent γ -glutamyl carboxylase according to claim 1, characterized in that: the increment of the fluorescence value calculated by the end-point method is as follows: detecting fluorescence once every a period of time by using a fluorescence detector, wherein the total detection time is 1-2 hours; activity of VKOR protein = fluorescence value F at 1 hour of reaction _T1 Background fluorescence value at the beginning of the reaction F _T0 。

7. A small molecule compound and its structural analogues screened by the method for screening a small molecule compound targeted to inhibit vitamin K-dependent γ -glutamyl carboxylase according to any one of claims 1 to 6.

8. Use of the small molecule compound of claim 7 and structural analogs thereof for the preparation of inhibitors targeting vitamin K-dependent γ -glutamyl carboxylase.

9. Use according to claim 8, characterized in that: the structural formula of the small molecule compound and the structural analogue thereof is as follows: