CN1049690C - Process for screening inhibitor of acetolactate synthetase and application - Google Patents

Abstract

The present invention relates to a screening method and the application of the inhibitor of acetolactate synthetase (ALS enzyme). The substance which is screened participates in the enzymatic reaction of the ALS enzyme in a test tube; if the substance is the needed enzyme inhibitor, the reaction rate is reduced, products are reduced, and then, the activity of the enzyme and the inhibitory activity of compounds can be quickly and accurately obtained by measuring the converted substances of reaction products. Thus, the screening process of the inhibitor of the ALS enzyme is realized. A novel blank concept and an optimal pH range are proposed, and four solvent systems are established; various samples to be detected can be dissolved, various compounds and derivatives thereof are detected, the lead compounds for developing high efficiency herbicides are discovered, and the IC50 value of concentration in the process of inhibition of the ALS enzyme is set.

Description

Process for screening inhibitor of acetolactate synthetase and application

The invention belongs to the screening method of enzyme inhibitors, particularly be process for screening inhibitor of acetolactate synthetase.

The top priority of initiative ultra-high efficiency low toxicity herbicide is to seek lead compound.Fast, find that exactly lead compound needs brand-new meaning, the screening method in vitro of molecular level.Selecting for use acetolactate synthestase (Acetolactate Synthase, abbreviation ALS) to make target, is the forward position focus of the current research and development of weedicide in the world.The ALS enzyme inhibitors is described as " milestone in the pesticide science development history ".Now confirmed sulfonylurea, imidazolone type, and triazolopyrimidines is the inhibitor of ALS enzyme.The ALS enzyme inhibitors of new texture type also continues to bring out.The at present domestic potted plant screening method that has general integral body to measure weeding activity, this method judge that the screening effect required time is long, generally take 10 to 14 days, look into hand dipping by sight and provide the result.Its resultant error is bigger, and sensitivity is low, and can not illustrate restraining effect mechanism effectively.By retrieval, U.S. Pat 5,141 discloses the weeding resistance plant of nucleic acid fragments coding for No. 870; US 5,206, disclose the oxygen sensitive electrode for No. 135 and surveyed the ALS enzymic activity.Two parts of patents ALS inhibitor sifting method of not touching upon.The document that has in the world discloses some data of relevant enzymatic reaction, but also be odd, as: 1. Shaner D.L., Anderson P.C., Plant Physiology, 76,545-546 (1984) plant physiology magazine is Holtzclaw W.D. 2., Journal of Bacteriology, 121,917-922 (1975) bacteriology magazine is Giuseppt Forlanl 3., Weed Science, 39,1. 553-557 (1991) weeds science magazine document has reported enzymatic reaction solution composition and concentration, K ₂HPO ₄20mM, Sodium.alpha.-ketopropionate 20mM, TPP 0.32mM, MuSO ₄0.5mM.What metal ion was used in the composition is mn ion, also need be centrifugal after reaction finishes, remove precipitation, and the existence of sulfate radical also influences the generation of product.Document 1., 2., the volume of 3. having introduced reaction soln is 0.5ml, this value is less, illustrates that each reactant of adding still less, causes that relative error can be bigger, easily misleads and suppresses active measurement result.Though the relation of pH value and optical density(OD) E when 2. document has provided enzymatic reaction does not have stable E value in the pH4-8 scope, not having best pH scope, can to cause measuring optical density(OD) E value inaccurate.So equal screening method of not complete report inhibitor of acetolactate synthetase in the above-mentioned data is still blank in the data that this method is at home and abroad published at present.Clearly propose in country's " eight or five " skill brainstorm project: " making great efforts to create new screening model ", our current task that Here it is.

The objective of the invention is:

It is complete to set up a cover, standard, the brand-new molecular level process for screening inhibitor of acetolactate synthetase that exsomatizes.Require this method to leave plant materials, in breadboard test tube, screened thing is participated in reaction under the katalysis of acetolactate synthestase, descend by enzyme ' s reaction speeding, it is the minimizing of product, fast, calculate the inhibition activity of screened thing exactly, thereby filter out the inhibitor of acetolactate synthestase.

Another purpose of the present invention is:

Use aforesaid method, fast, concentration Ic50 value during the acetolactate synthestase of definite inhibitor compound suppresses exactly, thus filter out the lead compound that might be developed as highy potent herbicide.

The object of the present invention is achieved like this:

1. select for use the quality acetolactate synthestase identical with quantity to make the catalyzer of enzymatic reaction, the reactant of enzymatic reaction is by the substrate of acetolactate synthestase, and coenzyme and damping fluid are formulated, and reaction process is:

Do not contain in the assaying reaction system and add in the enzyme ' s reaction speeding V0 of screened thing and the reaction system behind the screened thing, repressed enzyme ' s reaction speeding V1 determines to suppress active percentage ratio i% according to the two ratio, and its formula is: $i\%=(1-\frac{V1}{V0})\×100\%---\left(1\right)$ Speed of reaction V can represent that with E then formula (1) becomes with measuring the increasing amount representative that enzyme work is the product of time to time change: $i\%=(1-\frac{E1}{E0})\×100\%---\left(2\right)$ According to the E value of measuring, calculate the active i% of inhibition of screened thing, judge whether quality into inhibitor and inhibitor just can filter out inhibitor according to the active i% of inhibition, its screening process may further comprise the steps:

(1). at first select for use the quality acetolactate synthestase identical to make the catalyzer of enzymatic reaction with quantity;

(2). determine to dissolve fully a kind of solvent of screened thing, and prepare its solution;

(3). determine to carry out substrate in the reactant of enzymatic reaction, the composition and the concentration of coenzyme and buffering solution;

(4). enzyme is being arranged, and in the reaction solution that water and buffering solution are made into, (III IV) adds different materials and carries out enzymatic reaction, measures four kinds of product amount E values under the situation respectively for I, II by following four kinds of situations again;

In reaction solution, add the solvent of the screened thing of dissolving and when not adding screened thing:

The blank product amount of enzymatic reaction when I. not containing reactant in assaying reaction liquid EA;

II. add the enzymatic reaction product amount EB behind the reactant in the assaying reaction liquid;

After in reaction solution, adding screened thing solution:

The blank product amount of enzymatic reaction when III. not containing reactant in assaying reaction liquid EA1;

IV. add the enzymatic reaction product amount EB1 behind the reactant in the assaying reaction liquid;

(5). determine the active E0 of acetolactate synthestase, this activity equals to add the poor of the enzymatic reaction product amount EA of enzymatic reaction product amount EB when not having reactant behind the reactant, this value expression:

E0＝EB-EA

(6). determine the residual activity E1 of acetolactate synthestase when having screened thing to exist, this value expression:

E1＝EB1-EA1

(7). according to above-mentioned formula (2), calculate the inhibition activity of screened thing.

2. the blank product of described acetolactate synthestase enzymatic reaction means that acetolactate synthestase under the enzyme reaction condition of not having (adding) enzyme reaction thing, still constantly carries out enzymatic reaction, and the product of generation is blank.

3. the ratio of acetolactate synthestase enzyme is lived in being worth and is 5-12 μ M/mghr, substrate in the reactant of enzymatic reaction, and the composition of coenzyme and buffering solution and the condition and the product of concentration and enzymatic reaction are as follows:

Damping fluid: dipotassium hydrogen phosphate K ₂HPO ₄20mM

Substrate: Sodium.alpha.-ketopropionate CH ₃COCOOH 15.5mM

Coenzyme: magnesium chloride Mg Cl ₂0.4mM

Thiaminpyrophosphate TPP 0.4mM

Flavin adenine dinucleotide FAD 8 μ M

In above-mentioned reactant, add the solvent of 0.1ml and the enzyme of 0.5ml, be mixed with the reaction soln of 2.6ml,, under 39-40 ℃, carry out enzymatic reaction in a hour, generate the product acetylactis at normal pressure.

4. the optimal ph of the 3rd described acetolactate synthestase enzymatic reaction solution is at 6.9-7.5.

5. measure the acetolactic method of acetolactate synthestase enzymatic reaction product with surveying the method that enzyme is lived.

6. the 5th described process for screening inhibitor of acetolactate synthetase is characterized in that the method for described survey enzyme work can be used spectrophotometry, is to measure acetylactis is converted into the 3-oxobutanol under effect of sulfuric acid content, and reaction process is: Spectrophotometry in turn includes the following steps:

(1). in the enzymatic reaction solution of 2.6ml, add 0.05-0.25ml, the H of 6N ₂SO ₄

(2). with above-mentioned solution 59-60 ℃ of following constant temperature 15 minutes;

(3). add 0.5-1.5ml, 0.5% creatine (a kind of guanidine compound that contains);

(4). add 1-2ml again, 5% 1-alkali naphthols generates red complex;

(5). the mensuration liquor capacity is 3-7ml; At 59-60 ℃, constant temperature is 20 minutes under the normal pressure;

(6). take out solution, left standstill after shaking up 30-40 minute;

(7). with spectrophotometric determination E value, wavelength is 520nm, the 1cm cuvette.

7. dissolve the optional fresh water supply system of solvent of screened thing.

8. the solvent that dissolves screened thing can be selected acetone-inso system for use.

9. the solvent that dissolves screened thing can be selected the tetrahydrofuran (THF) system for use.

10. the solvent that dissolves screened thing can be selected the methyl-sulphoxide system for use.

11. above-mentioned the 1st described process for screening inhibitor of acetolactate synthetase, concentration IC during the ALS enzyme that can be used for determining screened inhibitor suppresses ₅₀Value, the lead compound when determining the initiative novel pesticide in view of the above, its determining step is as follows:

(1). select same a kind of inhibitor of different concns for use,, carry out enzymatic reaction respectively simultaneously by the described method of claim 1;

(2). according to the enzymatic reaction result, the enzyme of correspondence value alive when obtaining different concns, and then determine the residual activity percentage ratio of same inhibitor when different concns, i.e. 100%-i%;

(3). concentration and pairing residual activity percentage ratio with inhibitor are coordinate, draw sensitivity curve;

(4). finding out residual activity according to curve is 50% o'clock, and just suppressing active i% is 50% o'clock pairing concentration, concentration IC during the ALS enzyme that is this inhibitor suppresses ₅₀Value, the lead compound when determining the initiative novel pesticide in view of the above.

Method of the present invention is the basis that is determined as with enzyme work and enzyme work.Enzyme work refers to the ability of the chemical reaction that enzyme catalysis is certain, be actually measure one by enzyme the speed of catalytic chemical reaction.In enzymatic reaction, if add the catalytic capability that a kind of compound can suppress or block enzyme, the speed of chemical reaction will change, and we can select different technique means, measure the minimizing of substrate of time to time change or the increase of product, represent the speed of catalyzed reaction.Exist and the comparison that does not have two kinds of enzyme ' s reaction speedings under the condition by screened compound, calculate and suppress active, thereby filter out the active inhibitor compound of high inhibition.

Acetolactate synthestase is a kind of lyase, and its catalyzed reaction type is that decarboxylation and C-C generate.Substrate series is: (i) pyruvic acid, (ii) pyruvic acid and α-batanone acid; Product series is: (i) acetylactis, (ii) acetyl hydroxybutyric acid; The product series that is suitable for measuring through conversion is: (i) 3-oxobutanol, (ii) dimethyl diketone.

Acetolactate synthestase is at the coenzyme thiaminpyrophosphate, and the flavin adenine dinucleotide reaches under the existence of magnesium ion, and in suitable phosphate buffered saline buffer, the catalytic substrate pyruvic acid is the product acetylactis.The acetolactic content of time to time change has been represented this chemical reaction rate.Acetylactis can be used gas chromatography determination, also available spectrophotometry.If use spectrophotometry, then in the product acetylactis, add sulfuric acid, stop enzymatic reaction, acetylactis changes the 3-oxobutanol simultaneously into, and under a kind of effect that contains the guanidine compound creatine, 3-oxobutanol and 1-alkali naphthols form red network and thing.Determine that by network and thing absorption curves complex compound maximum absorption wavelength 520nm measures wavelength,, calculate product content, see Fig. 1 with optical density(OD) E value with 1cm cuvette colorimetric.X-coordinate is an absorbing wavelength among the figure, and unit is nm; Ordinate zou is optical density value E; Dotted line is the complex compound absorption curves; Solid line is blank.

Determining of enzymatic reaction condition:

1. determining that the enzymatic reaction temperature is that reaction product is relevant with the reaction times under 39-40 ℃ the situation, as if being as the criterion with 60 minutes, be about in the time of 30 minutes its 70%.

2. the pH value of enzymatic reaction system is seen Fig. 2 to the influence of enzyme ' s reaction speeding.X-coordinate is the pH value among the figure; Ordinate zou is optical density value E.

3. at the substrate q.s, when temperature and pH value were constant, reaction product and enzyme concn were linear, see Fig. 3.X-coordinate is an enzyme dosage among the figure, and unit is ml; Ordinate zou is optical density value E.

4. in enzyme concn, when temperature and pH value were constant, speed of reaction changed with concentration of substrate, and assigned maximum value V in a certain concentration, and the concentration of substrate during V/2 is Michaelis-Menton constant Km, and this experimental value is 3-4mM, sees Fig. 4.X-coordinate is a concentration of substrate among the figure, and unit is mM; Ordinate zou is optical density value E.

5. during enzyme ' s reaction speeding is measured, there is related parameter to establish a capital by experiment really and obtains.Comprise: add the vitriolic consumption during decarboxylation; The creatine consumption; 1-alkali naphthols consumption; The temperature of color reaction; Determining of network and thing steady time; With the working curve of standard substance 3-oxobutanol, can see Fig. 5 respectively successively; Fig. 6; Fig. 7; Fig. 8; Fig. 9; And Figure 10.Among Fig. 5, X-coordinate is 6N H ₂SO ₄Consumption, unit is ml; Ordinate zou is optical density value E.Among Fig. 6, X-coordinate is the consumption of 0.5% creatine, and unit is ml; Ordinate zou is optical density value E.Among Fig. 7, X-coordinate is the consumption of 5%1-alkali naphthols, and unit is ml; Ordinate zou is optical density value E.Among Fig. 8, X-coordinate is a solution color reaction temperature, and unit is ℃; Ordinate zou is optical density value E.Among Fig. 9, X-coordinate is the consumption of standard substance 3-oxobutanol, and unit is μ g; Ordinate zou is optical density value E; The a curve is the measured value of colour developing 55 timesharing; The b curve is the measured value of colour developing 75 timesharing; The c curve is the measured value of colour developing 25 timesharing.Among Figure 10, X-coordinate is the consumption of standard substance 3-oxobutanol, and unit is μ g; Ordinate zou is optical density value E.

The used acetolactate synthestase of the present invention is the patented product that Chemical Industry ﹠ Metallrygy Research Office of CAS produces, and other raw materials all can be buied on market.

Use method of the present invention, we have screened multiple screened compound, wherein, have found that two kinds have remarkable active compound Y-14 of inhibition and Y-29, same process for screening inhibitor of acetolactate synthetase of the present invention, the concentration IC in can being inhibited by sensitivity curve of using ₅₀Value, this will introduce in embodiment 11 and 12.

Positively effect of the present invention and advantage:

1. found isolated activity and grand close compound Y-14 and the Y-29 of chlorine sulphur with this screening method, under the identical situation of dosage, the grand inhibition of chlorine sulphur activity is 59%, and Y-14 is 52%, and Y-29 is 42%.The IC that chlorine sulphur is grand ₅₀Value (the inhibition activity is 50% o'clock the concentration of using) is 72 μ M, the IC of Y-14 ₅₀Value is 66 μ M, the IC of Y-29 ₅₀Value is 151 μ M.

The compound of these two kinds of novel structures is exactly promising lead compound owing to determined it is ALS enzyme inhibitors efficiently, might be developed to new highy potent herbicide through composition optimizes on this basis.

2. analysis speed is fast

Carry out owing to the intravital biochemical reaction of plant is moved on in the breadboard test tube, avoided the growth process of existing integral experiment, got rid of transmission, the interference of processes such as metabolism.Therefore, in 4 hours, a staff can screen 10-20 compound, and it is active to calculate inhibition.

3. few this screening method of amount of samples is only used 0.4 milligram screened compound, can provide the The selection result result.This will also can estimate smoothly to the low compound of those building-up reactions productive rates, need not to strengthen the raw material consumption, also save development cost.

4. applied widely

Present method provides can dissolve sample, does not influence four kinds of solvents of the enzymatic reaction and the system of mensuration again, and insoluble screened compound also can be dissolved, simultaneously, measured the inhibition activity of same screened compound under the different solvents system, its result is close, and illustration method is reliable.

5. good stability, accuracy is good

At the enzymatic reaction characteristics, the present invention proposes new blank notion, it is different from " reagent blank " notion of common sense.New blank notion is, enzyme is under the enzyme reaction condition of no enzyme reaction thing, and the product of the enzymatic reaction of carrying out is blank.Design program and deducted this partly blank, so calculated value can reflect the inhibition activity of inhibitor exactly.

In the method design, considered various influence stability, the factor of accuracy, such as the concentration of substrate value, the control of pH value of solution value, sulfuric acid consumption or the like during decarboxylation by a large amount of experiments, has been determined operational condition.Measuring method adopts spectrophotometry, has designed unique schedule of operation.1. considered that the optical density value that solvent itself causes changes, selected same solvent for use, compared under the same amount, therefore can offset the error that solvent effect causes, but 2. bucking-out system error, therefore, The ultimate results is better than general living survey method, and relative error is not more than 3-5%.

6. highly sensitive

Detect down as the product of rate of catalysis reaction foundation and to be limited to 0.2 microgram, the inhibition field of activity that can survey compound is 0-100%.

The present invention provides following graphic representation through a large amount of experiments:

Fig. 1. the absorption curves of complex compound;

Fig. 2. the relation curve of speed of reaction E and pH value of solution value;

Fig. 3. enzyme dosage is to optical density(OD) E value relation curve;

Fig. 4. the relation curve of speed of reaction E and concentration of substrate;

Fig. 5. add the relation curve of sulfuric acid consumption during the reaction product decarboxylation;

Fig. 6. the relation curve of E and 0.5% creatine consumption when reaction product is measured;

Fig. 7. the relation curve of E and 5%1-alkali naphthols consumption when reaction product is measured;

Fig. 8. the relation curve of E and solution color reaction temperature when reaction product is measured;

Fig. 9. the relation curve of E and complex compound steady time when reaction product is measured;

Figure 10. the working curve of standard substance 3-oxobutanol;

Figure 11. the sensitivity curve of compound TP;

Figure 12. the sensitivity curve that compound chlorine sulphur is grand;

Figure 13. the sensitivity curve of compound metsulfuronmethyl;

Figure 14. the sensitivity curve of compound Y-14;

Figure 15. the sensitivity curve of compound Y-29.

Below in conjunction with embodiment and accompanying drawing, the invention will be described further.

Embodiment 1: the screening verification experiment that compound chlorine sulphur is grand

(1) at first selects the catalyzer of doing enzymatic reaction than the value of living for the acetolactate synthestase of the 0.2-0.5ml of 5-12 μ M/mghr for use;

(2) solvent that can dissolve screened chlorine sulphur Longhua compound fully is a methyl-sulphoxide, and the grand sample of the chlorine sulphur of 10mg is dissolved in the 5ml dimethylsulfoxide solvent, is mixed with the solution that concentration is 2mg/ml.

(3) used substrate in the reactant of enzymatic reaction, the composition and the concentration of coenzyme and buffering solution are as follows:

Damping fluid: dipotassium hydrogen phosphate K ₂HPO ₄20mM

Substrate: Sodium.alpha.-ketopropionate CH ₃COCOOH 15.5mM

Coenzyme: magnesium chloride Mg Cl ₂0.4mM

Thiaminpyrophosphate TPP 0.4mM

Flavin adenine dinucleotide FAD 8 μ M

(4) according to the screening method program of table 1, prepare four test tubes, add 0.5ml enzyme, the K of 0.6ml 20mM to each test tube ₂HPO ₄Be made into enzymatic reaction liquid.

Table 1.ALS inhibitor sifting method program model (two parallel)

1 2 3

The screened thing K of project ₂HPO ₄Reactant enzyme H ₂O 39-40 ℃

20mM

Test tube (ml) is (ml) (ml) (ml) (branch) (ml)

#1 solvent 0.1 0.6 0.0 0.5 1.4 60

#2 solvent 0.1 0.6 1.0 0.5 0.4 60

#3 sample 0.1 0.6 0.0 0.5 1.4 60

#4 sample 0.1 0.6 1.0 0.5 0.4 60

Table 1. (continuing)

456 project H ₂SO ₄59-60 ℃ of room temperature of 59-60 ℃ of creatine 1-alkali naphthols placed E

6N 0.5% 5% 520nm test tubes (ml) (branch) are (ml) (branch) (branch) 1cm ware #1 0.2 15 1.0 2.0 20 35 EA#2 0.2 15 1.0 2.0 20 35 EB#3 0.2 15 1.0 2.0 20 35 EA1#4 0.2 15 1.0 2.0 20 35 EB1 (ml)

Six kinds of listed in the table 1 solution are formulated as follows:

1. screened compound solution: claim the 10mg compound to be dissolved in the water of 5ml, or acetone, or methyl-sulphoxide, or in the tetrahydrofuran (THF), concentration is 2mg/ml;

2.K ₂HPO ₄Solution: claim 0.4564g K ₂HPO ₄3H ₂O is dissolved in the 100ml water;

3. reactant solution: 1. K ₂HPO ₄: claim 0.9128g K ₂HPO ₄3H ₂O is dissolved in 20ml water;

2. Sodium.alpha.-ketopropionate: claim that 0.4401g is dissolved in 20ml water;

3. magnesium chloride: claim that 0.0203g is dissolved in 10ml water;

4. TPP: claim that 0.0461g is dissolved in 10ml water;

5. FAD: claim that 1.659mg is dissolved in 10ml water; In turn will be 1., 2., 3., 4., 5. pour in the 100ml volumetric flask, add water to scale;

4.6N H ₂SO ₄Solution: get dense H ₂SO ₄16.7ml slowly add in the entry, to 100ml;

5.0.5% creatine: claim that the 0.5g creatine is water-soluble, to 100ml;

6.5%1-alkali naphthols: claim the 1-alkali naphthols of 1.0g purifying to be dissolved in the NaOH solution of 2.5N to 20ml.

Press following I again, II, III, four kinds of situations preparations of IV:

At #1, add the dimethylsulfoxide solvent of 0.1ml in the #2 test tube and when chlorination sulphur is grand:

Add the water of 1.4ml in the I#1 test tube, liquor capacity is assigned to 2.6ml, actual measurement pH value is 7.2, carries out the enzymatic reaction of reactionless thing this moment, and its product amount is blank.Reaction conditions is: under normal pressure, 39-40 ℃, reaction was carried out one hour, generated the product acetylactis.According to the method for measuring enzyme ' s reaction speeding, promptly spectrophotometry is surveyed the content of this product, in turn includes the following steps:

1. in the enzymatic reaction solution of 2.6ml, add the H of 0.2ml 6N ₂SO ₄, stopped reaction also is converted into the 3-oxobutanol with acetylactis;

2. with above-mentioned solution 59-60 ℃ of following constant temperature 15 minutes;

3. the creatine that adds 1ml 0.5%;

4. the 1-alkali naphthols that adds 2ml 5% again, the mensuration solution of composition 5.8ml, promptly red network and thing solution;

5. at 59-60 ℃, constant temperature is 20 minutes under the normal pressure;

6. take out test tube, after shaking up, room temperature left standstill 35 minutes;

7. detect with spectrophotometer, wavelength is 520nm, with 1cm cuvette colorimetric, surveys the optical density value EA=0.429 of product.For obtaining exact value, do parallel laboratory test according to aforesaid method, get EA=0.433, get its mean value, EA=0.431 sees Table 2.

Add (3) described reaction solution 1.0ml in the II#2 test tube, the water that adds 0.4ml again, equally liquor capacity is assigned to 2.6ml, actual measurement pH value is 7.3, the enzymatic reaction of thing responds this moment, its reaction conditions is identical with the step and the I that survey product content, measures mean value EB=0.912, sees Table 2.

At #3, add (2) described chlorine sulphur Longhua compound solution of 0.1ml in the #4 test tube after:

Add the water of 1.4ml in the III#3 test tube, liquor capacity is assigned to 2.6ml, actual measurement pH value is 7.3, and the enzymatic reaction blank assay of reactionless thing is arranged under the grand existence of chlorine sulphur this moment.Its reaction conditions is identical with the step and the I that survey product content, measures mean value EA1=0.439, sees Table 2.

Add (3) described reactant 1.0ml in the IV#4 test tube, add the water of 0.4ml again, equally liquor capacity is assigned to 2.6ml, actual measurement pH value is 7.4, and other is identical with the said determination step, measures enzymatic reaction thing quantity behind the adding reactant, get its mean value EB1=0.636, see Table 2.

(5) determine the active E0 of acetolactate synthestase:

E0＝EB-EA＝0.912-0.431＝0.481

(6) the residual activity E1 of acetolactate synthestase when having that chlorine sulphur is grand to be existed:

E1＝EB1-EA1＝0.635-0.439＝0.197

(7) calculate the active i% of the grand inhibition of chlorine sulphur: $i\%=(1-\frac{E1}{E0})\×100\%=(1-\frac{0.197}{0.481})\×100\%=59\%$

Because of chlorine sulphur is grand is known efficient ALS enzyme inhibitors, and the result can verify the feasibility and the reliability of the inventive method thus.

The screened thing chlorine of the determination data table of table 2. embodiment 1-7 (each screened thing sample is 10mg) embodiment #1 #2 #3 #4 #5 #6 #7 sulphur swells metsulfuron-methyl TP Y-14 R-2 Li-2 Lu-5 molecular weight 357.3 381.4 302.0 438.0 305.3 332.0 394.0 concentration μ M 96.5 90.4 114.0 78.0 112.0 103.0 87.0

Diformazan tetrahydrochysene diformazan diformazan tetrahydrochysene solvent sulfoxide furans sulfoxide sulfoxide furans water acetone EA (single) 0.429 0.660 0.628 0.665 0.660 0.367 0.394

0.433 0.664 0.618 0.657 0.664 0.364 0.392EA (on average), 0.431 0.662 0.623 0.661 0.662 0.365 0.393EB (single) 0.908 0.885 1.067 1.088 0.885 0.890 0.699

0.916 0.894 1.095 0.999 0.894 0.872 0.690EB (on average), 0.912 0.889 1.081 1.004 0.889 0.881 0.694E0=EB-EA, 0.481 0.227 0.458 0.343 0.227 0.516 0.301EA1 (single) 0.431 0.663 0.628 0.660 0.655 0.364 0.402

0.446 0.667 0.626 0.668 0.665 0.368 0.396EA1 (on average), 0.439 0.665 0.627 0.664 0.660 0.366 0.399EB1 (single) 0.635 0.760 0.798 0.828 0.851 0.889 0.695

0.636 0.768 0.806 0.829 0.867 0.875 0.675EB1 (on average), 0.636 0.764 0.802 0.828 0.859 0.882 0.685E1=EB1-EA1 0.197 0.099 0.175 0.164 0.199 0.516 0.286 suppresses active % 59 56 62 52 12 05

Embodiment 2: the screening verification experiment of compound metsulfuronmethyl

(1) at first selects the catalyzer of doing enzymatic reaction than the value of living for the acetolactate synthestase of the 0.2-0.5ml of 5-12 μ M/mghr for use;

(2) solvent that can dissolve screened metsulfuronmethyl compound fully is a tetrahydrofuran (THF), and the metsulfuronmethyl sample of 10mg is dissolved in the 5ml tetrahydrofuran solvent, is mixed with the solution that concentration is 2mg/ml.

(3) used substrate in the reactant of enzymatic reaction, the composition and the concentration of coenzyme and buffering solution are as follows:

Damping fluid: dipotassium hydrogen phosphate K ₂HPO ₄20mM

Substrate: Sodium.alpha.-ketopropionate CH ₃COCOOH 15.5mM

Coenzyme: magnesium chloride Mg Cl ₂0.4mM

Thiaminpyrophosphate TPP 0.4mM

Flavin adenine dinucleotide FAD 8 μ M

(4) according to the screening method program of table 1, prepare four test tubes, add 0.5ml enzyme, the K of 0.6ml 20mM to each test tube ₂HPO ₄Be made into enzymatic reaction liquid.Again by following four kinds of situations preparation:

At #1, add the tetrahydrofuran solvent of 0.1ml in the #2 test tube and when not adding metsulfuronmethyl:

Add the water of 1.4ml in the I#1 test tube, liquor capacity is assigned to 2.6ml, actual measurement pH value is 6.9, carries out the enzymatic reaction of reactionless thing this moment, and its product amount is blank.Reaction conditions is: under normal pressure, 39-40 ℃, reaction was carried out one hour, generated the product acetylactis.According to the method for measuring enzyme ' s reaction speeding, promptly spectrophotometry is surveyed the content of this product, in turn includes the following steps:

1. in the enzymatic reaction solution of 2.6ml, add the H of 0.2ml 6N ₂SO ₄, stopped reaction also is converted into the 3-oxobutanol with acetylactis;

2. with above-mentioned solution 59-60 ℃ of following constant temperature 15 minutes;

3. the creatine that adds 1ml 0.5%;

4. the 1-alkali naphthols that adds 2ml 5% again, the mensuration solution of composition 5.8ml, promptly red network and thing solution;

5. at 59-60 ℃, constant temperature is 20 minutes under the normal pressure;

6. take out test tube, after shaking up, room temperature left standstill 35 minutes;

7. detect with spectrophotometer, wavelength is 520nm, with 1cm cuvette colorimetric, surveys the optical density value EA=0.660 of product.For obtaining exact value, do parallel laboratory test according to aforesaid method, get EA=0.664, get its mean value, EA=0.662 sees Table 2.

Add (3) described reaction solution 1.0ml in the II#2 test tube, the water that adds 0.4ml again, equally liquor capacity is assigned to 2.6ml, actual measurement pH value is 7.0, the enzymatic reaction of thing responds this moment, its reaction conditions is identical with the step and the I that survey product content, measures mean value EB=0.889, sees Table 2.

At #3, add (2) described metsulfuronmethyl compound solution of 0.1ml in the #4 test tube after:

Add the water of 1.4ml in the III#3 test tube, liquor capacity is assigned to 2.6ml, actual measurement pH value is 7.0, and the enzymatic reaction blank assay of reactionless thing is arranged under the metsulfuronmethyl existence this moment.Its reaction conditions is identical with the step and the I that survey product content, measures mean value EA1=0.665, sees Table 2.

Add (3) described reactant 1.0ml in the IV#4 test tube, add the water of 0.4ml again, equally liquor capacity is assigned to 2.6ml, actual measurement pH value is 7.1, and other is identical with the said determination step, measures enzymatic reaction thing quantity behind the adding reactant, get its mean value EB1=0.764, see Table 2.

(5) determine the active E0 of acetolactate synthestase:

E0＝EB-EA＝0.889-0.662＝0.227

(6) the residual activity E1 of acetolactate synthestase when having metsulfuronmethyl to exist:

E1＝EB1-EA1＝0.764-0.665＝0.099

(7) the active i% of the inhibition of calculating metsulfuronmethyl: $i\%=(1-\frac{E1}{E0})\×100\%=(1-\frac{0.099}{0.227})\×100\%=56\%$

Because of metsulfuronmethyl is known efficient ALS enzyme inhibitors, the result also can verify the feasibility and the reliability of the inventive method thus.

Embodiment 3: the screening verification experiment of compound triazolopyrimidine derivative TP

(1) at first selects the catalyzer of doing enzymatic reaction than the value of living for the acetolactate synthestase of the 0.2-0.5ml of 5-12 μ M/mghr for use;

(2) solvent that can dissolve screened compound fully is a methyl-sulphoxide, and the TP sample of 10mg is dissolved in the 5ml dimethylsulfoxide solvent, is mixed with the solution that concentration is 2mg/ml.

(3) used substrate in the reactant of enzymatic reaction, the composition and the concentration of coenzyme and buffering solution are as follows:

Damping fluid: dipotassium hydrogen phosphate K ₂HPO ₄20mM

Substrate: Sodium.alpha.-ketopropionate CH ₃COCOOH 15.5mM

Coenzyme: magnesium chloride Mg Cl ₂0.4mM

Thiaminpyrophosphate TPP 0.4mM

Flavin adenine dinucleotide FAD 8 μ M

(4) according to the screening method program of table 1, prepare four test tubes, add 0.5ml enzyme, the K of 0.6ml 20mM to each test tube ₂HPO ₄Be made into enzymatic reaction liquid.Again by following four kinds of situations preparation:

At #1, add the dimethylsulfoxide solvent of 0.1ml in the #2 test tube and when not adding TP:

Add the water of 1.4ml in the I#1 test tube, liquor capacity is assigned to 2.6ml, actual measurement pH value is 7.5, carries out the enzymatic reaction of reactionless thing this moment, and its product amount is blank.Reaction conditions is: under normal pressure, 39-40 ℃, reaction was carried out one hour, generated the product acetylactis.According to the method for measuring enzyme ' s reaction speeding, promptly spectrophotometry is surveyed the content of this product, in turn includes the following steps:

1. in the enzymatic reaction solution of 2.6ml, add the H of 0.2ml 6N ₂SO ₄, stopped reaction also is converted into the 3-oxobutanol with acetylactis;

2. with above-mentioned solution 59-60 ℃ of following constant temperature 15 minutes;

3. the creatine that adds 1ml 0.5%;

4. the 1-alkali naphthols that adds 2ml 5% again, the mensuration solution of composition 5.8ml, promptly red network and thing solution;

5. at 59-60 ℃, constant temperature is 20 minutes under the normal pressure;

6. take out test tube, after shaking up, room temperature left standstill 35 minutes;

7. detect with spectrophotometer, wavelength is 520nm, with 1cm cuvette colorimetric, surveys the optical density value EA=0.628 of product.For obtaining exact value, do parallel laboratory test according to aforesaid method, get EA=0.618, get its mean value, EA=0.623 sees Table 2.

Add (3) described reaction solution 1.0ml in the II#2 test tube, the water that adds 0.4ml again, equally liquor capacity is assigned to 2.6ml, actual measurement pH value is 7.4, the enzymatic reaction of thing responds this moment, its reaction conditions is identical with the step and the I that survey product content, measures mean value EB=1.081, sees Table 2.

At #3, add (2) described TP compound solution of 0.1ml in the #4 test tube after:

Add the water of 1.4ml in the III#3 test tube, liquor capacity is assigned to 2.6ml, actual measurement pH value is 7.5, the enzymatic reaction blank assay of reactionless thing is arranged under the TP existence at this moment.Its reaction conditions is identical with the step and the I that survey product content, measures mean value EA1=0.627, sees Table 2.

Add (3) described reactant 1.0ml in the IV#4 test tube, add the water of 0.4ml again, equally liquor capacity is assigned to 2.6ml, actual measurement pH value is 7.4, and other is identical with the said determination step, measures enzymatic reaction thing quantity behind the adding reactant, get its mean value EB1=0.802, see Table 2.

(5) determine the active E0 of acetolactate synthestase:

E0＝EB-EA＝1.081-0.623＝0.458

(6) the residual activity E1 of acetolactate synthestase when having TP to exist:

E1＝EB1-EA1＝0.802-0.627＝0.175

(7) the active i% of the inhibition of calculating TP: $i\%=(1-\frac{E1}{E0})\×100\%=(1-\frac{0.175}{0.458})\×100\%=62\%$

Because of TP is known efficient ALS enzyme inhibitors, the result also can verify the feasibility and the reliability of the inventive method thus.

Embodiment 4: the screening of artificial-synthetic compound Y-14

The Y-14 that gets 10mg is dissolved in the 5ml dimethylsulfoxide solvent, and being mixed with concentration is the Y-14 solution of 2mg/ml.Step according to identical with embodiment 1 adds identical reactant with under identical condition, carries out enzymatic reaction, and when measuring enzyme ' s reaction speeding, the solution of adding is respectively: the H of 0.05ml 6N ₂SO ₄, the creatine of 1.5ml 0.5%, the 1-alkali naphthols of 1ml 5%; Add water to the mensuration solution of 5.80ml, network and thing time of repose are 30 minutes.The optical density value of reaction product is shown in Table 2 under four kinds of situations, and it suppresses active i%=52%.

Embodiment 5: the screening of artificial-synthetic compound R-2

The R-2 that gets 10mg is dissolved in the 5ml tetrahydrofuran solvent, and being mixed with concentration is the R-2 solution of 2mg/ml.Step according to identical with embodiment 1 adds identical reactant with under identical condition, carries out enzymatic reaction, and when measuring enzyme ' s reaction speeding, the solution of adding is respectively: the H of 0.05ml 6N ₂SO ₄, the creatine of 0.5ml 0.5%, the 1-alkali naphthols of 1ml 5%; Add water to the mensuration solution of 5.80ml, network and thing time of repose are 30 minutes.The optical density value of reaction product is shown in Table 2 under four kinds of situations, and it suppresses active i%=12%.

Embodiment 6: the screening of artificial-synthetic compound Li-2

The Li-2 that gets 10mg is dissolved in the 5ml water, and being mixed with concentration is the Li-2 solution of 2mg/ml.Step according to identical with embodiment 1 adds identical reactant with under identical condition, carries out enzymatic reaction, and when measuring enzyme ' s reaction speeding, the solution of adding is respectively: the H of 0.25ml 6N ₂SO ₄, the creatine of 0.5ml0.5%, the 1-alkali naphthols of 1.5ml 5%; Add water to the mensuration solution of 5.80ml, network and thing time of repose are 30 minutes.The optical density value of reaction product is shown in Table 2 under four kinds of situations, and it suppresses active i%=0%, illustrates that the Li-2 compound does not suppress active.

Embodiment 7: the screening of artificial-synthetic compound Lu-5

The Lu-5 that gets 10mg is dissolved in the 5ml acetone, and being mixed with concentration is the Lu-5 solution of 2mg/ml.Below measure according to embodiment 1 method fully.The optical density value of reaction product is shown in Table 2 under four kinds of situations of gained, and it suppresses active i%=5%, illustrates that compound L u-5 only has faint inhibition activity.

Embodiment 8: the IC that determines compound TP ₅₀Value

1. according to the screening method program of table 1, carry out, at first measure when not having reactant, add the dimethylsulfoxide solvent of 0.1ml, the K of 0.6ml 20mM successively by table 3 row order according to the method for embodiment 1 ₂HPO ₄, the ALS enzyme of 0.5ml, the water of 1.4ml is mixed with the reaction solution of 2.6ml, carries out enzymatic reaction one hour at 39-40 ℃ of following constant temperature, surveys enzyme ' s reaction speeding with spectra photometric method, adds the H of 0.2ml 6N that is: ₂SO ₄, 59-60 ℃ of following constant temperature 15 minutes adds the creatine of 1ml 0.5%, adds the 1-alkali naphthols of 2ml 5%, 59-60 ℃ of following constant temperature 20 minutes, and room temperature was placed 35 minutes, and at 520nM, surveying blank product amount EA1 value with the 1cm cuvette is 0.627, sees Table 3.

The parameter of table 3. embodiment 8 (TP) and determination data table examination reaction K ₂HPO ₄Reaction enzymes H ₂O 39-H ₂SO ₄40 ℃ of 6N of 59-creatine pipe concentration 20mM liquid 60 ℃ No. 0.5% (μ M) are (ml) (ml) (ml) (branch) (ml) 0 0.1ml of (ml) (branch) (ml) _Solvent0.6 0 0.5 1.4 60 0.2 15 1.01 0 0.6 1.0 0.5 0.4 60 0.2 15 1.02 57.09 0.6 1.0 0.5 0.4 60 0.2 15 1.03 114.18 0.6 1.0 0.5 0.4 60 0.2 15 1.04 171.27 0.6 1.0 0.5 0.4 60 0.2 15 1.05 228.36 0.6 1.0 0.5 0.4 60 0.2 15 1.0

Table 3. (continuing) examination 1-alkali naphthols 59-room temperature E is residual, and live 5% 60 ℃ of pipes are placed (%) 0 2.0 20 35 EA1=0.6271,2.0 20 35 EB0=1.085 E0=EB0-EA1=0.458,1,002 2.0 20 35 EB1=0.848 E1=EB1-EA1=0.221,483 2.0 20 35 EB2=0.802 E2=EB1-EA1=0.175,384 2.0 20 35 EB3=0.778 E3=EB1-EA1=0.151,325 2.0 20 35 EB4=0.744 E4=EB1-EA1=0.117 25 number (ml) (dividing) (dividing) (520nm, 1cm)

According to the method described above, measure the enzymatic reaction product amount EB value of TP under five kinds of different concns situations again, EB0, EB1, EB2, EB3, EB4 is respectively 1.085,0.848, and 0.802,0.778 and 0.744.The poor E0 of they and EA1 (0.627), 1,2,3,4 are respectively 0.458,0.221, and 0.175,0.151 and 0.117.Its corresponding TP concentration is respectively 0 μ M, 57.09 μ M, and 114.18 μ M, 171.27 μ M and 228.36 μ M see Table 3.

2. with residual activity percentage ratio formula: $r\%=\frac{\mathrm{<figure-callout\; id="0"\; label="E"\; filenames="C9510982300282.png,C9510982300293.png"\; state="\{\{state\}\}">\; <figure-callout\; id="1"\; label="E"\; filenames="C9510982300281.png,C9510982300321.png"\; state="\{\{state\}\}">E</figure-callout>\; </figure-callout>}0,1,2,3,4}{\mathrm{<figure-callout\; id="0"\; label="E"\; filenames="C9510982300282.png,C9510982300293.png"\; state="\{\{state\}\}">E</figure-callout>}0}\&times;100\%$ Calculate the residual activity percentage ratio of TP under five kinds of concentration, respectively be 100%, 48%, 38%, 32% and 25%.

3. the concentration value with TP is an X-coordinate, is ordinate zou with corresponding residual activity percentage ratio r%, draws sensitivity curve, sees Figure 11.

4. finding out residual activity from sensitivity curve is that the concentration value of 50% o'clock pairing TP is 57 μ M, is concentration IC in the inhibition of TP ₅₀Value.

Embodiment 9: determine the IC that compound chlorine sulphur is grand ₅₀Value

1. according to the screening method program of table 1, carry out, at first measure when not having reactant, add the dimethylsulfoxide solvent of 0.1ml in the following order successively, the K of 0.6ml 20mM according to the method for embodiment 1 ₂HPO ₄, the ALS enzyme of 0.5ml, the water of 1.4ml is mixed with the reaction solution of 2.6ml, carries out enzymatic reaction one hour at 39-40 ℃ of following constant temperature, surveys enzyme ' s reaction speeding with spectra photometric method, adds the H of 0.2ml 6N that is: ₂SO ₄, 59-60 ℃ of following constant temperature 15 minutes adds the creatine of 1ml 0.5%, adds the 1-alkali naphthols of 2ml 5%, 59-60 ℃ of following constant temperature 20 minutes, and room temperature was placed 35 minutes, and at 520nM, surveying blank product amount EA1 value with the 1cm cuvette is 0.458.

According to the method described above, measure chlorine sulphur again and swell enzymatic reaction product amount EB value under five kinds of different concns situations, EB0, EB1, EB2, EB3, EB4 is respectively 0.839,0.687, and 0.635,0.592 and 0.553.The poor E0 of they and EA1 (0.458), 1,2,3,4 are respectively 0.381,0.229, and 0.177,0.134 and 0.095.The grand concentration of its corresponding chlorine sulphur is respectively 0 μ M, 48.25 μ M, 96.50 μ M, 144.75 μ M and 193.00 μ M.

2. with residual activity percentage ratio formula: $r\%=\frac{\mathrm{<figure-callout\; id="0"\; label="E"\; filenames="C9510982300282.png,C9510982300293.png"\; state="\{\{state\}\}">\; <figure-callout\; id="1"\; label="E"\; filenames="C9510982300281.png,C9510982300321.png"\; state="\{\{state\}\}">E</figure-callout>\; </figure-callout>}0,1,2,3,4}{\mathrm{<figure-callout\; id="0"\; label="E"\; filenames="C9510982300282.png,C9510982300293.png"\; state="\{\{state\}\}">E</figure-callout>}0}\&times;100\%$ Calculate chlorine sulphur and swell residual activity percentage ratio under five kinds of concentration, respectively be 100%, 60%, 46%, 35% and 24%.

3. being X-coordinate with the grand concentration value of chlorine sulphur, is ordinate zou with corresponding residual activity percentage ratio r%, draws sensitivity curve, sees Figure 12.

4. finding out residual activity from sensitivity curve is that 50% o'clock grand concentration value of pairing chlorine sulphur is 72 μ M, is concentration IC in the grand inhibition of chlorine sulphur ₅₀Value.

Embodiment 10: the IC that determines the compound metsulfuronmethyl ₅₀Value

1. according to the screening method program of table 1, carry out, at first measure when not having reactant, add the dimethylsulfoxide solvent of 0.1ml in the following order successively, the K of 0.6ml 20mM according to the method for embodiment 1 ₂HPO ₄, the ALS enzyme of 0.5ml, the water of 1.4ml is mixed with the reaction solution of 2.6ml, carries out enzymatic reaction one hour at 39-40 ℃ of following constant temperature, surveys enzyme ' s reaction speeding with spectra photometric method, adds the H of 0.2ml 6N that is: ₂SO ₄, 59-60 ℃ of following constant temperature 15 minutes adds the creatine of 1ml 0.5%, adds the 1-alkali naphthols of 2ml 5%, 59-60 ℃ of following constant temperature 20 minutes, and room temperature was placed 35 minutes, and at 520nM, surveying blank product amount EA1 value with the 1cm cuvette is 0.683.

According to the method described above, measure the enzymatic reaction product amount EB value of metsulfuronmethyl under five kinds of different concns situations again, EB0, EB1, EB2, EB3, EB4 is respectively 0.938,0.835, and 0.782,0.742 and 0.740.The poor E0 of they and EA1 (0.683), 1,2,3,4 are respectively 0.255,0.152, and 0.099,0.059 and 0.057.Its corresponding metsulfuronmethyl concentration is respectively 0 μ M, 45.20 μ M, 90.40 μ M, 135.60 μ M and 180.80 μ M.

2. with residual activity percentage ratio formula: $r\%=\frac{\mathrm{<figure-callout\; id="0"\; label="E"\; filenames="C9510982300282.png,C9510982300293.png"\; state="\{\{state\}\}">\; <figure-callout\; id="1"\; label="E"\; filenames="C9510982300281.png,C9510982300321.png"\; state="\{\{state\}\}">E</figure-callout>\; </figure-callout>}0,1,2,3,4}{\mathrm{<figure-callout\; id="0"\; label="E"\; filenames="C9510982300282.png,C9510982300293.png"\; state="\{\{state\}\}">E</figure-callout>}0}\&times;100\%$ Calculate the residual activity percentage ratio of metsulfuronmethyl under five kinds of concentration, respectively be 100%, 59%, 38%, 23% and 22%.

3. the concentration value with metsulfuronmethyl is an X-coordinate, is ordinate zou with corresponding residual activity percentage ratio r%, draws sensitivity curve, sees Figure 13.

4. finding out residual activity from sensitivity curve is that the concentration value of 50% o'clock pairing metsulfuronmethyl is 58 μ M, is concentration IC in the inhibition of metsulfuronmethyl ₅₀Value.

Embodiment 11: the IC that determines lead compound Y-14 ₅₀Value

1. according to the screening method program of table 1, carry out, at first measure when not having reactant, add the dimethylsulfoxide solvent of 0.1ml in the following order successively, the K of 0.6ml 20mM according to the method for embodiment 1 ₂HPO ₄, 0.5ml the ALS enzyme, the water of 1.4ml is mixed with the reaction solution of 2.6ml, carries out enzymatic reaction one hour at 39-40 ℃ of following constant temperature, surveys enzyme ' s reaction speeding with spectra photometric method, adds the H of 0.2ml 6N that is: ₂SO ₄, 59-60 ℃ of following constant temperature 15 minutes adds the creatine of 1ml 0.5%, adds the 1-alkali naphthols of 2ml 5%, 59-60 ℃ of following constant temperature 20 minutes, and room temperature was placed 35 minutes, and at 520nM, surveying blank product amount EA1 value with the 1cm cuvette is 0.664.

According to the method described above, measure the enzymatic reaction product amount EB value of Y-14 under five kinds of different concns situations again, EB0, EB1, EB2, EB3, EB4 is respectively 1.007,0.881, and 0.828,0.812 and 0.716.The poor E0 of they and EA1 (0.664), 1,2,3,4 are respectively 0.343,0.217, and 0.164,0.148 and 0.097.Its corresponding Y-14 concentration is respectively 0 μ M, 39.36 μ M, 78.72 μ M, 118.09 μ M and 157.44 μ M.

2. with residual activity percentage ratio formula: $r\%=\frac{\mathrm{<figure-callout\; id="0"\; label="E"\; filenames="C9510982300282.png,C9510982300293.png"\; state="\{\{state\}\}">\; <figure-callout\; id="1"\; label="E"\; filenames="C9510982300281.png,C9510982300321.png"\; state="\{\{state\}\}">E</figure-callout>\; </figure-callout>}0,1,2,3,4}{\mathrm{<figure-callout\; id="0"\; label="E"\; filenames="C9510982300282.png,C9510982300293.png"\; state="\{\{state\}\}">E</figure-callout>}0}\&times;100\%$ Calculate the residual activity percentage ratio of Y-14 under five kinds of concentration, respectively be 100%, 63%, 47%, 43% and 28%.

3. the concentration value with Y-14 is an X-coordinate, is ordinate zou with corresponding residual activity percentage ratio r%, draws sensitivity curve, sees Figure 14.

4. finding out residual activity from sensitivity curve is that the concentration value of 50% o'clock pairing Y-14 is 66 μ M, is concentration IC in the inhibition of Y-14 ₅₀Value.

Embodiment 12: the IC that determines lead compound Y-29 ₅₀Value

1. according to the screening method program of table 1, carry out, at first measure when not having reactant, add the dimethylsulfoxide solvent of 0.1ml in the following order successively, the K of 0.6ml 20mM according to the method for example I ₂HPO ₄, the ALS enzyme of 0.5ml, the water of 1.4ml is mixed with the reaction solution of 2.6ml, carries out enzymatic reaction one hour at 39-40 ℃ of following constant temperature, surveys enzyme ' s reaction speeding with spectra photometric method, adds the H of 0.2ml 6N that is: ₂SO ₄, 59-60 ℃ of following constant temperature 15 minutes adds the creatine of 1ml 0.5%, adds the 1-alkali naphthols of 2ml 5%, 59-60 ℃ of following constant temperature 20 minutes, and room temperature was placed 35 minutes, and at 520nM, surveying blank product amount EA1 value with the 1cm cuvette is 0.628.

According to the method described above, measure the enzymatic reaction product amount EB value of Y-29 under five kinds of different concns situations again, EB0, EB1, EB2, EB3, EB4 is respectively 1.006,0.960, and 0.846,0.805 and 0.754.The poor E0 of they and EA1 (0.628), 1,2,3,4 are respectively 0.378,0.332, and 0.218,0.177 and 0.126.Its corresponding Y-29 concentration is respectively 0 μ M, 50.65 μ M, 101.30 μ M, 151.95 μ M and 202.60 μ M.

2. with residual activity percentage ratio formula: $r\%=\frac{\mathrm{<figure-callout\; id="0"\; label="E"\; filenames="C9510982300282.png,C9510982300293.png"\; state="\{\{state\}\}">\; <figure-callout\; id="1"\; label="E"\; filenames="C9510982300281.png,C9510982300321.png"\; state="\{\{state\}\}">E</figure-callout>\; </figure-callout>}0,1,2,3,4}{\mathrm{<figure-callout\; id="0"\; label="E"\; filenames="C9510982300282.png,C9510982300293.png"\; state="\{\{state\}\}">E</figure-callout>}0}\&times;100\%$ Calculate the residual activity percentage ratio of Y-29 under five kinds of concentration, respectively be 100%, 88%, 58%, 47% and 33%.

3. the concentration value with Y-29 is an X-coordinate, is ordinate zou with corresponding residual activity percentage ratio r%, draws sensitivity curve, sees Figure 15.

4. finding out residual activity from sensitivity curve is that the concentration value of 50% o'clock pairing Y-29 is 151 μ M, is concentration IC in the inhibition of Y-29 ₅₀Value.

Claims (6)

1. process for screening inhibitor of acetolactate synthetase, it is characterized in that: select for use acetolactate synthestase to make the catalyzer of enzymatic reaction, the reactant of enzymatic reaction is formulated by damping fluid, substrate and coenzyme, and reaction process is expressed as:

Do not contain the enzyme ' s reaction speeding V1 that adds in the enzyme ' s reaction speeding V0 of screened thing and the reaction system behind the screened thing in the assaying reaction system, according to the two ratio, determine to suppress active percentage ratio i%, its formula is: $i\%=(1-\frac{V1}{V0})\&times;100\%---\left(1\right)$ Speed of reaction V can represent that with E then formula (1) becomes with measuring the increasing amount representative that enzyme work is the product of time to time change: $i\%=(1-\frac{E1}{E0})\&times;100\%---\left(2\right)$ According to the E value of measuring, calculate the active i% of inhibition of screened thing, judge whether quality into inhibitor and inhibitor just can filter out inhibitor according to the active i% of inhibition, its screening process may further comprise the steps:

(1). at first select for use than the value 5-12 μ m/mghr that lives, volume is the catalyzer that the acetolactate synthestase of 0.2-0.5ml is done enzymatic reaction;

(2). determine to dissolve fully a kind of solvent of screened thing, and be mixed with the solution that concentration is 2mg/ml;

(3). the composition and the concentration in reaction system of the contained damping fluid of reactant, substrate and coenzyme of determining to carry out enzymatic reaction is as follows:

Damping fluid: dipotassium hydrogen phosphate K ₂HPO ₄20mM

Substrate: Sodium.alpha.-ketopropionate CH ₃COCOOH 15.5mM

Coenzyme: magnesium chloride Mg Cl ₂0.4mM

Thiaminpyrophosphate TPP 0.4mM

Flavin adenine dinucleotide FAD 8 μ M

(4). in four test tubes, add the enzyme of 0.5ml and the K of 0.6ml 20mM respectively earlier ₂HPO ₄, (III IV) adds or does not add solvent, screened thing solution, reactant and an amount of H for I, II by following four kinds of situations again ₂O is mixed with the reaction soln system of 2.6ml, and the pH value of this system is 6.9-7.5, at normal pressure, and 39-40 ℃, carry out enzymatic reaction in 1 hour, generate the product acetylactis, expression formula is as follows:

Measure product amount E value respectively by following four kinds of situations:

In reaction system, add the solvent 0.1ml of the screened thing of dissolving and when not adding screened thing:

The blank product amount of enzymatic reaction when I. not containing reactant in assaying reaction system EA;

II. add the enzymatic reaction product amount EB behind the 1ml reactant in the assaying reaction system;

When in reaction system, adding the screened thing solution of 0.1ml:

The blank product amount of enzymatic reaction when III. not containing reactant in assaying reaction system EA1;

IV. add the enzymatic reaction product amount EB1 behind the 1ml reactant in the assaying reaction system;

(5). determine the active E0 of acetolactate synthestase, this activity equal to add behind the reactant enzymatic reaction product amount EB with do not have the poor of reaction volume EA, this value expression:

E0＝EB-EA

(6). determine the active E1 of acetolactate synthestase when having screened thing to exist, this activity equals to add the poor of the enzymatic reaction product amount EA1 of enzymatic reaction product amount EB1 when not having reactant behind the reactant, this value expression:

E1＝EB1-EA1

(7) according to the formula in the claim 1 (2), calculate the inhibition activity of screened thing, spectrophotometry is selected in enzyme assay for use, is to measure the product acetylactis is converted into the 3-oxobutanol under effect of sulfuric acid content, and reaction process is expressed as: Spectrophotometry is surveyed the E value and is in turn included the following steps:

1.. in the enzymatic reaction solution system of 2.6ml, add 0.05-0.25ml, the H of 6N ₂SO ₄

2.. with above-mentioned solution 59-60 ℃ of following constant temperature 15 minutes;

3.. add 0.5-1.5ml, 0.5% creatine;

4.. add 1-2ml again, 5% 1-alkali naphthols generates red complex;

5.. the mensuration liquor capacity is 3-7ml; At 59-60 ℃, constant temperature is 20 minutes under the normal pressure;

6.. take out solution, shake up the back room temperature and placed 30-40 minute;

7.. with spectrophotometric determination E value, wavelength is 520nm, the 1cm cuvette.

2. process for screening inhibitor of acetolactate synthetase according to claim 1, it is characterized in that the blank product of described acetolactate synthestase enzymatic reaction means that acetolactate synthestase is under the enzyme reaction condition of no enzyme reaction thing, still constantly carry out enzymatic reaction, the product of generation is blank.

3. process for screening inhibitor of acetolactate synthetase according to claim 1 is characterized in that the optional fresh water supply system of solvent of the screened thing of described dissolving.

4. process for screening inhibitor of acetolactate synthetase according to claim 1 is characterized in that the solvent of the screened thing of described dissolving can be selected acetone-inso system for use.

5. process for screening inhibitor of acetolactate synthetase according to claim 1 is characterized in that the solvent of the screened thing of described dissolving can be selected the tetrahydrofuran (THF) system for use.

6. process for screening inhibitor of acetolactate synthetase according to claim 1 is characterized in that the solvent of the screened thing of described dissolving can be selected the methyl-sulphoxide system for use.

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