GB2523616A

GB2523616A - Use of quinolinone compound as hCBS enzyme inhibitor

Info

Publication number: GB2523616A
Application number: GB1417893.3A
Authority: GB
Inventors: Jing Yu; Fang Wu; Yueyang Zhou
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2013-10-09
Filing date: 2014-10-09
Publication date: 2015-09-02
Anticipated expiration: 2034-10-09
Also published as: GB2523616B; GB201417893D0; CN104546849A; CN104546849B

Abstract

The use of quinolinone compound as human cystathionine Î²-synthase (hCBS) enzyme inhibitors, particularly the use of the compound 7-amino-2-hydroxy-5-imino-6,7-dihydroquinolin-8(5H)-one as an hCBS enzyme inhibitor. The small molecule compound is 5,7-diamino quinolinone compound, the IC50 of which for in vitro hCBS enzyme assay is 20 ÂµM. Such small molecule compound can be used as a drug tool for studying H2S signaling pathways, and as a lead compound for developing the drugs for treating the H2S related diseases, such as circulatory shock, stroke, Down's syndrome, etc. In another aspect, a method for inhibiting in vitro hCBS using7-amino-2-hydroxy-5-imino-6,7-dihydroquinolin-8(5H)-one, said method involving the detection of H2S. In another aspect, a method for inhibiting the activity of the intracellular hCBS enzyme using7-amino-2-hydroxy-5-imino-6,7-dihydroquinolin-8(5H)-one, the method involving the detection of H2S.

Description

Use of quinolinone compound as hCBS enzyme inhibitor

Field of Invention

The present invention relates to the field of biomedicine, particularly relates to the use of a human cystathionine 3-synthase enzyme inhibitor.

Description of Related Arts

The human endogenous H2S gas molecule involves and regulates a plurality of physiological and pathological processes, and is an important regulatory molecule of signal transduction pathways. Under many pathological conditions of colon cancer, neurodegenerative disease, ischernic stroke, low blood pressure, high blood pressure.

pancreatitis and the like, the endogenous H2S content is found to have abnormal change.

Thus, at present, specific inhibitor or activator of the endogenous 1-125 signal is required to use as a molecular probe tool to further illustrate the physiological functions of H2S. and as a small molecular drug lead to treat the 1-12S-related diseases.

CBS cystathionine f3-synthase) is a PLP-dependent (pyñdoxal-5 -phosphate -dependent) enzyme. i.e., vitamin B6-dependent enzyme. In human body. CBS catalyzes the specific substrates of L-Cysteine and L-Homocysteine to generate endogenous H2S gas.

In circulatory shock, stroke, Down's syndrome and cancer patients or model animals, excessive H2S and/or cthiancemcnt of CBS activity is found, thus, hCBS (human CBS) has been considered as a potential target to treat the H2S-related diseases, such as circulatory shock, stroke, Down's syndrome and tumor and the like.

The known hCBS small molecular inhibitors have, not only thw affinity to enzymes. but also low selectivity, since they also have inhibiting effect on other vitamin B6-dependent enzymes. Thus, it needs to screen a specific inhibitor to used as a "drug tool to verify the found biological effects and signal pathway effect of 1-12S, and meanwhile.

to used as a lead compound for new drug discovery R&D.

Currently. it generally adopts two methods for the discovery of enzyme inhibitors, i.e.. biological screening am! rational drug design. Target-based rational drug design refers to: study on the targets affected by drugs. and searching for novel, proper drugs for treating some specific diseases. Normally, the discovery of one target can lead to a design of a new class of drugs. and is paid more attentions in the field of new drug research.

When deeply understanding the enzyme structure, especially the structure when it complexes with a specific inhibitor, wouk! produce a method for identifying the binding sites of such enzyme, and obtain the conformations of an enzyme/inhibitor compound and sensitive residues in such enzyme, while such knowledge is crucial for the drug design and optimization method.

The 3D crystal structure of the hCBS pure enzyme has heen reported, by which has provided a certain structural foundation for the design of its targeting inhibitor, while the amino acid residues related to the substrate-bonded are still unclear yet.

Therefore, those skilled in the art dedicated to studying the inhibitors of hCBS. as well as the use of such inhibitors as drug lead compounds for studying the treatment of the H2S-rclated diseases, such as circulatory shock, stroke, Downs syndrome, etc.

Summary of the Present Invention

The abbreviations being used in the present invention are as follows: hCBS refers to: human cystathionine f3-synthase; DTNB refers to: 5. 5-Dithio bis-(2-nitrobenzoic acid); Tris-HC1 refers to: Tris(l ydroxymethyl)aminomethane hydrochloride; EDTA refers to: Ethylene Diamine Tetraacetic Acid: L-Cys refers to: L-Cysteine; D, L-HCys refers to: D.L-Homocysteine; SAM refers to: 5-adenosyl methionine: PLP refers to: pyridoxal-5'-phosphate;

I

NEAA refers to non-essential amino acids; 7-amino-2-liydroxy-5-imino-&7-dihydroquinolin-8(5H)-one.

i.e., 7-amino-2-hydroxy-5-irnino-6,7-dihydroquinolin-8(5H)-one; 2-arnino-4-imino-6-rnethyl-4,4a-dihydropteridin-7-ol, i.e., 2-amino-4-imino-6-me(hyl-4,4a-dihydropteridin-7-ol: 7-(-aminobenzyl)-1-imino-7.9b-dthydro-1H-pyrro1o[3,2-f]quinazo1in-3-amine, i.e.. 7-(4-aminobenzyl)-l-imino-7,9b-dihydro-1H-pyrrolo[3.2-f] quinazolin-3-amine; HepG2 cell refers to: a human liver carcinoma cell line.

The enzymatic reaction vessel mentioned in the present invention enables to house a detection vessel of H2S gas. i.e., the gas detection vessel is able to be arranged in the enzymatic reaction vessel, such that the whole H2S gas generated by the enzymatic reaction is able to diffuse into (he detection system.

The present invention discloses a use of a quinolinone compound as an hCBS enzyme inhibitor, thc compound is 7-amino-2-hydroxy-5-imino-6,7-dihydroquinolin-8(5H)-one, with the structure as shown in formula (I), and the molecular weight is 191.19: H NH2 (I).

In the present invention. Compd. 1 is used to refer to 7-amino-2-hydroxy-5-imino-6.7-dihydroquinolin-8(5H)-one; Compd.8 is used to refer to 2-amino-4-imino-6-methyl-4,4a-dihydropteridin-7-oI; Compd.9 is used to refer to 7-(4-aminobenzyl)-l-irnino-7,9b-dihydro-1H-pyrrolo[3,2-f] quinazolin-3-arnine.

The present invention discloses a method for inhibiting the enzymatic activity of in vitro hCBS. which method comprises thc following steps: step 1. preparing buffer solution: prepare deioniLed water in a enzymatic reaction vessel with Tris-HC1 at a concentration of 50 mM, PLP at a concentration of 100 jiM.

hCBS-413 at a concentration of 50-500 nM, L-Cys at a concentration of 4 mM, D,L-HCys at a concentration of 4 mM, of which the p1-I value is 7.6 to 9.0; step 2. preparing enzymatic reaction mixture: compound Compd. 1 is added into the prepared buffer solution in step 1, respectively, to prepare enzymatic reaction mixtures with different concentrations, to form die enzymatic reaction system; step 3, preparing detection system of H2S gas: DTNB solution of 50 1IL is added into a detection vessel of H2S gas, wherein the DTNB so'ution is: deionized water with DTNB at a concentration of 3 OOpM, Tris-HCI at a concentration of 262 mM. EDTA at a concentration of 13 mM, of which the pH value is 8.9.

to form the detection system of ItS gas; the gas detection vessel is placed in the enzymatic reaction vessel, such that the I-12S gas generated by the enzymatic reaction enables to diffuse into the gas detection system; step 4, incubation process: the eniymatic reaction vessel is sealed hy a microplate sealer. then is incubated at 37 C for 60 minutes; stepS, detect the applicative effect of compound Compd.l being used as an hCBS inhibitor: the light absorption of the detection system of I-12S gas at 413 nM is detected byinicroplate reader.

Preferably, in the above method for inhibiting hCBS, said pH value in step 1 is 8.6.

Preferably, in the above method Icr inhibiting hCBS. said hCBS-413 in step I is at a concentration of 100 nM.

Preferably, in the ahove method for inhibiting hCBS. said compound Cornpd. I in step 2 is at a concentration of 0 to 400 tM.

Preferably, in the above method for inhibiting hCBS, said compound Cornpd. 1 in step 2 is at a concentration of 4 1iM.

On the other ham!, the present. invention discloses a use of compound Compd. 1 as an intracellular hCBS enzyme inhibitor, comprising the following steps: step 1, after cultivating human HepG2 in amino acid nutrient liquid for one day, the compound Cornpd. 1 is added into the amino acid nutrient liquid, to conduct co-incubation; then, cells are washed by cold Tris-HC1 buffer solution twice, and are collected by a cell brush; the collected cells in the splitting buffer solution is frozen by liquid nitrogen, followed by be unfrozen at 37 C for 2 minutes. aM such freeze and unfreeze are repeated for three times; alter that, the lysate is centrifuged by a centrifuge for 1 hour, then the supernatant is coflected as 1-IepG2 cell solution to carry out the next experiment; the concentration of the Tris-HC1 buffer solution is 50 mM, and the pH value of the Tris-HC1 buffer solution is 8.6.

step 2, the obtained HepG2 cdl solution in step 1 is added into the enhyrnatic reaction vessel, followed by be added with deionized water of 20 jjL with PLP at a concentration of 100 tM, SAM at a concentration of 200 1iM. and deionized water with L-Cys at a concentration of 4 mM, D. L-HCys at a concentration of 4 mM. to form the intracellular enzymatic reaction system.

step 3, preparing detection system of H2S gas: DTNB solution of 50 tL is added into a detection vessel of H2S gas. wherein the DTNB solution is: deionized water with DTNB at a concentration of 300 1jM, Tris-HCI at a concentration of 262 mM, EDTA at a concentration of 13 mM. of which the pH value is 8.9, to form the detection system of H2S gas; the gas detection vessel is placed in the enzymatic reaction vessel, such that the H2S gas generated by the enzymatic reaction enables to dilfuse into the gas detection system; step 4, incubation process: the enzymatic reaction vessel is sealed by a microplate scaler, then is incubated at 37'C for 60 minutes; step 5. detect the applicative effect of compound Compd. 1 being used as an intracellular hCBS enzyme inhibitor: the light absorption of the detection system of H2S gas at 413 nM is detected by mieroplate reader.

Further, when the compound Compd.l is used as the intracellular hCBS enzyme inhibitor, the final concentration of added Compd.1 in step 1 is lO1jM or S1iM.

Further, the quinolinone compound of structural fornufla (I) in the present invention can be used as a drug for trealing circulatory shock, stroke. Down's syndrome, etc. The present invention studies the structure-activity relationship of the compound Compd.1 analogues as an hCBS enzyme inhibitor, with the result as shown in table 1. From table 1, one can see that, both Compd. 1 and Compd.8 arc effective inhibitors for hCBS, while Compd.9 has no activity for inhibiting hCBS. This is probably caused by the lack of hydroxyl group in 2-loci. thus, indicating that the hydroxyl group is crucial for maintaining the inhibitory effect of hCBS enzymatic reaction of such compound.

Table 1

Compound Structure In vitro 1C50 OtM); ____________ _____________________ hCBS -4 13 Compd. I 4 Compd.8 25 Mt2 Compd9 >400 3E Hereinafter. the conception, concrete structure and the generated technical ellects ci the present invention will be further described in combination with drawings, to adequately understand the ohjects. features and effects of the invention.

Brief Description of the Drawings

Figure I is concentration-activity curves ci compounds Compd.1 Compd.S Compd.9; Figure 2 is an active histogram of thc intracellular hCBS enzyme inhibited by the compound Compd.l at different concentrations.

Detailed Description of the Preferred Embodiments

The materials used in the present invention are as foflows: 1.1 Source of cells HepG2 cells refer to a human liver carcinoma cell line, and are purchased from Cell Resource Center, Shanghai institutes for biological sciences, CAS.

1.2 main reagents L-cysteine Sangon company DTNB Sangon company D, L-homoeysteine TCI Shanghai L-Cys Sangon All other drugs or reagents are purchased from Sigma-Aldrich.

UltraClear microplate sealer is from Platemax PCR-TS, USA.

The adopted experimental method of the present invention is as follows: Embodiment 1, expression and purification of the expression and purification method of the source truncated hCBSA414-551 (hCBS-413) According to the described methods in Frank, N., Arch Biochem Biophys 470, 64-72 (2008), Oliveriusova, J., J Biol Chem 277, 48386-94 (2002) or Janosik, M. et al., Acta Crystallogr D Biol Crystallogr 57, 289-91 2OO1), thc hCBSt414-551 hCBS-4l3) gene is, firstly. cloned to the fusional expression vector pGEX-KG of glutathione S-transferase (GST), then is overexpressed, and finally, perform affinity purification by using OST-agarose column.

The sequence table, SEQ ID No: 1, is the amino acid sequence of hCBS enzyme.

Thc usc of the compound Compd.1 as an hCBS enzyme inhibitor: Embodiment 2, detecting the inhibition ratio of different concentrations of compounds Compd. 1 with rcspcct to the activity of hCBS cnzymc: step I, preparing huller solution: prepare deionized water in a enzymatic reaction vessel with Tris-I-ICI at a concentration of 50 mM, PLP at a concentration of 100 jiM, hCBS-413 at a concentration of lOOnM, L-Cys at a concentration o14 mM, D,L-HCys at a concentration of 4 mM, of which the pH value is 8.6; step 2, preparing enzymatic reaction mixture: compound Conipd. 1 is added into the prepared buffer solution in step 1, respectively, to prepare enzymatic reaction mixtures with compound Compd. 1 at concentrations of 400tM 200jiM, lOOjiM, 50tM, 25jiM 125jiM 62pM, 3J25pM. 1.56jiM 0.78pM, 0391iM, 01951.iM, 0.097jjM 0048jiM, 0.024jiM O.Ol2pIvL 0.OO6jiM, respectively, to form the enzymatic reaction system; step 3, preparing detection system of lbS gas: DTNB solution of 50 1IL is added into a detection vessel of H2S gas, wherein the DTNB solution is: deionized water with DTNB at a concentration of 300 ilvI, Tris-HC1 at a concentration of 262 mM. EDTA at a concentration of 13 mM. of which the pH value is 8.9, to form the detection system of H2S gas; the gas detection vessel is placed in the enzymatic reaction vessel, such that the H2S gas generated by the enzymatic reaction enables to diffuse into the gas detection system; step 4, incubation process: the enzymatic reaction vessel is sealed by a microplate sealer, then is incubated at 37 C for 60 minutes; step 5. detect the applicative effect of compound Compd.1 being used as an hCBS inhibitor: the light absorption of the detection system of H2S gas at 413 nM is detected by microplatc reader.

The experiment is conducted twice separatdy, the activity of the negative control group (2°DMSO, concentration in volume) is defined as 100%.

The obtained concentration-activity curves is shown in figure 1.

Embodiment 3, detecting the inhibition ratio of different concentrations of compounds Compd.8 with respect to the activity of hCBS enzyme: Step 1, preparing buffer solution: prepare deionized water in a enzymatic reaction vessel with Tris-HC1 at a concentration of 50 mM, PLP at a concentration of 100 jiM.

hCBS-413 at a concentration of 100 nM. L-Cys at a concentration of 4 mM. D,L-HCys at a concentration of 4 mM, of which the p1-I va'ue is 8.6; step 2, preparing enzymatic reaction mixture: compound Compd.8 is added into the prepared buffer solution in step I, respectively, to prepare enzymatic reaction mixtures with compound Compd.8 at concentrations of 4001iM, 200jiM, lOOjiM, SOjiM, 25jiM, 125jiN'l, 62ptvl, 3.l25jiN'l, l.5GjiN'l, 0.78ptvl, respectively, to form the enzymatic reaction system; step 3, preparing detection system of H2S gas: DTNB solution of 50 jiL is addcd into a detection vessel of l-12S gas, wherein the DTNB solution is: deionized watcr with DTNB at a concentration of 300 1jM, Tris-HCI at a concentration of 262 mM, EDTA at a concentration of 13 mM, of which the pH value is 8.9.

to form the detection system of H25 gas; the gas detection vessel is placed in the enzymatic reaction vessel, such that the H2S gas generated by the enzymatic reaction enables to diffuse into the gas detection system; step 4, incubation process: the enzymatic reaction vessel is sealed by a microplate sealer, then is incubated at 37 C for 60 minutes; step 5, detect the applicative effect of compound Compd.8 being used as an hCBS inhibitor: the light absorption of the detection system of 1-125 gas at 413 nM is detected byrnireroplate reader.

The experiment is conducted twice separately, the activity of the negative control group (2°DMSO, concentration in volume) is defined as 100%.

The obtained concentration-activity curve is shown in figure 1.

Embodiment 4. detecting the inhibition ratio of different concentrations of compounds Compd.9 with respect to the activity of hCBS enzyme; Step 1, preparing buffer solution: prepare deionized water in a enzymatic reaction vessel with Tris-HC1 at a concentration of 50 mM, PLP at a concentration of lOO1iM, hCBS-413 at a concentration of 100 nM, L-Cys at a concentration of 4 mM, D,L-1-lCys at a concentration of 4 mM. pH value is 8.6; step 2, preparing enzymatic reaction mixture: compound Compd.9 is added into the prepared buffer solution in step 1, respectively, to prepare the enzymatic reaction mixture with compound Compd.9 at concentrations of 4001iM, 2001iM, 100pM, 50pM, 25p1v1, l2.SplVl, 6.2pM, 3.125p1v1, l.56j11'vl, 0.78p1v1, respectively, to form the enzymatic reaction system; step 3. preparing detection system of H2S gas: DTNB solution of 50 1IL is added into a detection vessel of H25 gas, wherein the DTNB solution is: deioniLed water with DTNB at a concentration of 300 pM, Tris-HC1 at a concentration of 262 mM. EDTA at a concentration of 13 mM, of which the pH value is 8.9.

to form the detection system of H2S gas; the gas detection vessel is placed in the enzymatic reaction vessel, such that thc H2S gas gcncratcd by the enzymatic reaction enables to diffuse into the gas detection system; stcp 4, incubation process: the enzymatic reaction vessel is sealed by a microplate sealer, then incubated at 37 t for 60 minutes; step 5, detect the applicative effect of compound Compd.9 being used as an hCBS inhibitor: the light absorption of the detection system of H2S gas at 413 nM is detected bymicroplate reader.

The experiment, is conducted twice separately, the activity of the negative control group (2°ADMSO, concentration in volume) is defined as 100%.

The obtained concentration-activity curve is shown in figure 1.

Embodiment 5. detecting the inhibitory effect of Coinpd.l with respect to hCBS enzyme in HepG2 cells: step 1. in CO2 gas with temperature at 37°C and humidity at 5%, the human l-lepG2 is cultivated in amino acid nutrient liquid MEM (Sangon company) (comprising lx nonessential amino acid. 10% fetal bovine serum and 1% (w/v) penicillin and streptomycin) in the cell culture 6-wall plate (density of -4x 105 per wall) coated with poly-D-lysine.

After incubating for one day, thc control group (blank DMSO solution) or the compound Compd.l is added into the above amino acid nutrient liquid, to form the cell culture media with the containing compound Compd. 1 at a final conccntration of lO1iM. 5tM, and thcn is co-incubated at 37 C. for 8 hours. After that, cells are washed by cold Tris-IICI buffer solution (50 niM, p1-1=8.6) twice, and are collected by a cell brush. The collected cells in the splitting buffer solution of 50 1iL (Tris-HCI of 50 pL, p1-I value is 8.6) is frozen by liquid nitrogen, followed by be unfrozen at 37 C for 2 minutes, and such frccze and unfreeze are repeated for three times. Then. (he lysate is centrifuged by a centrifuge at 4°C, with 11,000 rpm. for 1 hour, then the supernatant is collected. The protein concentration of the collected supernatant was detected by a BCA kit (pierce, USA), i.e., the next experiment is conductcd for thc HcpG2 ccli solution, in which thc activity of thc hCBS enzyme is inhibited by the compound Compd.1.

stcp 2, the obtaincd HcpG2 ccli solution, in which the activity of thc hCBS enzyme is inhibited by the compound Compd. 1, of 20 jiL in step 1 is added into the enzymatic reaction vessel, followed by he added with deionized water of 2OpL with PLP at a concentration of 100 jiM, SAM at a concentration of 200 tM, and deionized water of 10j.iL with L-Cys at a concentration of 4 mM. D.L-l-lCys at a concentration of 4 mM, to foim intracellular enzymatic reaction system.

step 3, preparing detection system of l-12S gas: DTNB solution of 50 RL is added into a detection vessel of H2S gas, wherein the DTNB solution is: deionized water with DTNB at a concentration of 300pM, Tris-l-ICI at a concentration of 262 mM. EDTA at a concentration of 13 mM, of which the pH value is 8.9.

to form the detection system of H2S gas; the gas detection vessel is placed in the enzymatic reaction vessel, such that thc H2S gas gcncratcd by the enzymatic reaction enabics to diffuse into the gas detection system; step 4, incubation process: the enzymatic reaction vessel is sealed by a microplate sealei; then incubate at 37 C for 60 minutes; step 5. detect the applicative effect of compound Compd.1 being used as an hCBS inhibitor: the light absorption of the detection system of H2S gas at 413 nM is detected bymicroplate reader; Taking the activity of the obtained intracelliflar hCBS enzyme by adding the control group (i.e., blank DMSO solution) as 100%, the activity of the intracellular hCBS enzyme inhibited by the compound Compd.1 at different concentrations is shown in figure 2.

The preferred embodiments of the present invention are described in detail as above. It is understood that, those skilled in the art may make many modifications and variations in accordance with the conception of the prcsent invention. Therefore, those technical solutions obtained by logical analysis, ratiocination and limited experiment of those skilled in the art in accordance with the conception of the invention on the basis of the prior art, should be within the scope of protection defined by the claims.

SEQUENCE LISTING

<ItO> Shanghai Jiaotong University <120> Use of quinolinone compound as hCBS enzyme inhibitor <130> P530906GB <160> 1 <170> Patentln version 13 <210> 1 <211> 551 <212> PRT <213> horno sapiens <400> 1 Met Pro Ser Clii Thir Pro Gin Aia Giu Val Giy Pro Thr Giy Cys Pro 10 15 His Arg Ser Gly Pro His Ser Ala Lys Gly Ser Len Gin Lys Gly Ser 25 30 Pro Glu Asp Lys Glu Ala Lys Glu Pro Leu Trp TIe Arg Pro Asp Ala 40 45 Pro Ser Anj Cys Thr Trp Gin Leu Giy Arg Pro Aia Ser Giu Ser Pro 55 60 His His His Thr Ala Pro Ala Lys Sec Pro Lys lie Len Pro Asp lie 70 75 80 Leu Lys Lys Tie Gly Asp Thr Pro Met Vai Arg He Asn Lys lie Giy 90 95 Lys Lys Phe Giy Len Lys Cys Giu Leu Leu Aia Lys Cys Giu Phe Phe 105 110 Asn Aia Giy Giy Ser Vai Lys Asp Arg He Ser Leu Arg Met Tie Glu 120 125 Asp Aia Gin Arg Asp Gly Thr Leu Lys Pro Giy Asp Thr lie lie Gin 135 140 Pro Thr Sec Giy Asn Thr Gly lie Giy Leu Ala Leu Ala Ala Ala Val 150 155 160 Arg Giy Tyr Arg Cys lie He Vai Met Pro Giu Lys Met Ser Ser Giu 170 175 Lys Val Asp Va! Leu Arg Ala Leu Giy Ala Giu ile Vai Arg Thr Pro 185 190 Thr Asn Ala Arg Plie Asp 5cr Pro Glu 5cr His Val Gly Val Ala Trp 200 205 Arg Leo Lys Asn GilL lie Pro Asn 5cr His lie Leo Asp Gin Tyr Arg 210 215 220 Asn Ala 5cr Asn Pro Leu Ala His Tyr Asp Thr Tlir Ala Asp Glu lie 225 230 235 240 Leu Gin Gin Cys Asp Giy Lys Leu Asp Met Leu Val Ala Ser Val Gly 245 250 255 Thr Gly Gly Thr ile Thr Giy lie Ala Arg Lys Leo Lys Glu Lys Cys 260 265 270 Pro Giy Cys Arg Tie Tie Gly Val Asp Pro Ghi Giy Ser lie Leu Ala 275 280 285 GIn Pro Glu Gln Len Asn GIn Thr Gln GIn Thr Thr Tyr GIn Val Glu 290 295 300 City Tie Gly Tyr Asp Phe Tie Pro Thr Vat Len Asp Arg Thr Vat Vat 305 310 315 320 Asp Lys Trp Phe Lys Ser Asn Asp Glu Giu Ala Phe Thr Phe Ala Arg 325 330 335 Met Leu Ile Ala Clii GIn Gly Leu Leu Cys Gly Gly 5cr Ala Gly 5cr 340 345 350 Thr Val Ala Val Ala Vai Lys Ala Ala Gin Giu Leu Gin Giu Giy Gin 355 360 365 Arg Cys Val Val lie Len Pro Asp Set Val Arg Asn Tyr Met Thr Lys 370 375 380 Phe Leu 5cr Asp Arg Trp Met Leu Gin Lys Gly Phe Leu Lys Giu Glu 385 390 395 400 Asp Len Thr Gin Lys Lys Pro Trp Trp Tip His Len Arg Val Ghi Gin 405 410 415 Len (fly Len Ser Ala Pro Len Thr Val Len Pro Tlir TIe Thr Cys Gly 420 425 430 His Thr lie Gb ile Leo Arg GilL Lys Giy Phe Asp Gin Ala Pro Vai 435 440 445 Va! Asp Gin Ma Gly Val Tie Len Gly Met Val Thr Leu Gly Asn Met 450 455 460 Leu Ser Ser Len Len Ala Giy Lys Vai Gin Pro 5cr Asp Gin Vai Giy 465 470 475 480 Lys Val lie Tyr Lys Gin Phe Lys Gin lie Arg Leu Thr Asp Thr Leu 485 490 495 Giy Arg Len 5cr His lie Len Glu Met Asp His Phe Ala Leu Vai Val 500 505 510 His Gin Ghi He Gin Tyr His 5cr Thr Giy Lys 5cr 5cr Gin Arg Gin 515 520 525 Met Va! Phe Giy Vai Vai Thr Aia He Asp Leu Leu Asu Phe Vai Aia 530 535 540 Aia Gin Gin Arg Asp Gin Lys 545 550

Claims

Claims: 1. Use of a quinolinone compound as an hCBS enzyme inhibitoi; the compound is 7-amino-2-hydroxy-5-imino-6,7-dihydroquinolin-8(5H)-one, with the structure as shown in formula (I), and the molecular weight is 191.19: HO NH2 (1).
2. A mcthod for inhibiting the enzymatic reaction of in vitro hCBS. which method compr sing the following steps: step 1, preparing buffer solution: prepare deionized water in a enzymatic reaction vessel with Tris-HC1 at a concentration of 50mM, PLP at a concentration of 100 1iM. hCBS-413 at a concentration of 50-500 nM, L-Cys at a concentration of 4 mM, D,L-HCys at a concentration of 4 mM, of which the pH value is 7.6 to 9.0; step 2, preparing enzymatic reaction mixture: compound 7-amino-2-hydroxy-5-imino-6,7-dihydroquinoHn-8H)-one is added into the prepared huller so'ution in step 1, respectively, to prepare enzymatic reaction mixtures with different concentrations, to fomt the enzymatic reaction system; step 3. preparing detection system of lbS gas: DTNB solution of 50 pL is added into a detection vessel of H2S gas, wherein the DTNB solution is: deionized water with DTNB at a concentration of 300 jiM, Tris-HC1 at a concentration of 262 mM, EDTA at a concentration of 13 mM. of which the pH value is 8.9, to form the detection system of H2S gas; the gas detection vessel is placed in the enzymatic reaction vessel, such that the H2S gas generated by the enzymatic reaction enables to diffuse into the gas detection system; step 4, incubation process: the enzymatic reaction vessel is sealed by a niicroplate scaler, then is incubated at 37 C for 60 minutes; step 5, detect the applicative effect of the compound 7-amino-2-hydroxy-5-imino-6,7-dihydroquinolin-8(5H)-one being used as an hCBS inhibitor: the light absorption olthe detection system of H2S gas at 413 nM is detected hymicroplate reader.
3. The method according to claim 2, wherein, the pH value in step I is 8.6.
4. The method according to claim 2, wherein, the hCBS-413 in step 1 is at a concentration of l00nM.
5. The method according to claim 2, wherein, the compound 7-amino-2-hydroxy-5-imino-6.7-dihydroquinolin-8(H)-onc in stcp 2 is at a concentration of 0-400 iM,
6. The method according to claim 2, wherein, the compound 7-amino-2-hydroxy-5-imino-6,7-dihydroquinofin-8(511)-one in step 2 is at a concentration of 41tM
7. A method for inhibiting the activity of the intracellular hCBS enzyme, which method comprising the following steps: step I, after cultivating human HepG2 in amino acid nutrient liquid for one day. the compound 7-arnino-2-hydroxy-5-imino-6,7-dihydroquinolin-8(SH)-one is added into the amino acid nutrient liquid, to conduct co-incubation; then, cells are washed by cold Tris-HC1 buffer scAution twice, and are collected by a cell brush; the collected cells in the splitting buffer so'ution is frozen by liquid nitrogen, followed by be unfrozen at 37 C for 2 nunutes, and such freeze and is unfreeze are repeated for three times; after that, the lysate is centrifuged by a centrifuge for 1 hour. then the supernatant is collected as HepG2 cell solution to carry out the next experiment; the concentration of the Tris-HCI buffer solution is 50 mM. and the pH value of the Tris-HCI huller solution is 8.6; step 2, the obtained HepG2 cell solution in step 1 is added into the enzymatic reaction vessel, followed by be added with deionized water of 20 jiL with PLP at a concentration of 100 tM, SAM at a concentration of 200 jM, and deionized water with L-Cys at a concentration of 4 mM, D. L-HCys at a concentration of 4 mM, to form intracellular enzymatic reaction system; step 3, preparing detection system of l-12S gas: DTNB solution of 50 pE is added into a detection vessel of H2S gas, wherein the DTNB solution is: deionized water with DTNB at a concentration of 300 tM, Tris-HC1 at a concentration of 262 mM, EDTA at a concentration of 13 mM. of which the pH value is 8.9. to form the detection system of H2S gas; the gas detection vessel is placed in the enzymatic reaction vessel, such that the H2S gas generated by the enzymatic reaction enables to diffuse into the gas detection system; step 4, incubation process: (he enzymatic reaction vessel is sealed by a microplate sealer, then is incubated at 37 t for minutes; step 5, detect the applicative effect of the compound 7-amino-2-hydroxy-5-imino-6.7-dihydroquinolin-8(5H)-one is used as an intracellular hCBS enzyme inhibitor: the light absorption of the detection system of I-12S gas at 413 nM is detected hymicroplate reader.
8. The method according to claim 7, wherein, the added 7-amino-2-hydroxy-5-imino-6,7-dihydroquinolin-8(5H)-one in step 1 is at a final concentration of 10, or 5MM.
9. Use of the quinolinone compound according to claim 1 in the manufacture of a medicament for the treatment of circulatory shock, stroke, Downs syndrome.