CN109632773B

CN109632773B - Screening method of dihydrolipoic acid succinyltransferase inhibitor

Info

Publication number: CN109632773B
Application number: CN201910014582.1A
Authority: CN
Inventors: 杨松; 陈彪; 龙青素; 赵永亮; 吴元元; 薛伟
Original assignee: Guizhou University
Current assignee: Guizhou University
Priority date: 2019-01-08
Filing date: 2019-01-08
Publication date: 2021-11-12
Anticipated expiration: 2039-01-08
Also published as: CN109632773A

Abstract

The invention relates to a screening method of a dihydrolipoic acid succinyltransferase inhibitor. Comprising screening dihydrolipoic acid succinyltransferase inhibitors in vitro and in vivo conditions. Firstly, pretreating total protein or recombinant dihydrolipoic acid succinyltransferase or living cells by an inhibitor to be screened, then treating a reaction system by a probe capable of marking the dihydrolipoic acid succinyltransferase, and coupling Biotin-N through click chemistry reaction₃And then imaging analysis is carried out by a streptavidin blot method. The establishment of the method has important significance for screening the dihydrolipoic acid succinyltransferase inhibitor.

Description

Screening method of dihydrolipoic acid succinyltransferase inhibitor

Technical Field

The invention relates to the field of biochemistry, relates to a screening method of a dihydrolipoic acid succinyltransferase inhibitor, and particularly relates to an in vitro and living screening method of a dihydrolipoic acid succinyltransferase inhibitor.

Background

Dihydrolipoic acid succinyltransferase (DLST), which is a core component of an alpha-ketoglutarate dehydrogenase complex (alpha-ketoglutarate dehydrogenase complex), plays a key role in catalyzing the conversion of alpha-ketoglutarate to succinyl-CoA, and is an important component of the tricarboxylic acid cycle (TCA)^[1-3]. Studies have shown that the DLST protein is closely related to the occurrence of various diseases, such as Alzheimer's disease^[4-6]Leukemia and leukemia^[7]Cardiovascular diseases^[8]And the DLST protein is an important disease treatment target, and the DLST inhibitor becomes a potential drug related to disease treatment. Currently, there are few highly potent and highly selective DLST inhibitors, which have led to great challenges in functional studies of DLST.

In recent years, CPI613 as an alpha-ketoglutarate dehydrogenase complex inhibitor has been used in clinical trials for the treatment of leukemia and non-small cell lung cancer^[9-10]. CPI613 is a lipoic acid analog that inhibits DLST activity by competitively binding to DLST with lipoic acid, but CPI613 also inhibits the activity of pyruvate dehydrogenase complex, which lacks selectivity, and thus there is a need to develop efficient and specific DLST inhibitors. However, due to the lack of an effective DLST inhibitor screening platform, the development of DLST inhibitors is still very difficult at present, so that the establishment of a high-efficiency DLST inhibitor screening method has very important significance for the development and functional research of DLST inhibitors.

The background art documents cited in the present application are as follows:

[1]Yang,L.；Shi,Q.；Ho,D.J.；Starkov,A.A.；Wille,E.J.；Xu,H.；Chen,H.L.；Zhang,S.；Stack,C.M.；Calingasan,N.Y.；Gibson,G.E.；Beal,M.F.Mice deficient in dihydrolipoyl succinyl transferase show increased vulnerability to mitochondrial toxins.Neurobiol.Dis.,2009,36,320-330.

[2]Starkov,A.A.An update on the role of mitochondrialα-ketoglutarate dehydrogenase in oxidative stress.Mol.Cell.Neurosci.,2013,55,13-16.

[3]Guo,H.；Madzak,C.；Du,G.；Zhou,J.Mutagenesis of conserved active site residues of dihydrolipoamide succinyltransferase enhances the accumulation ofα-ketoglutarate in Yarrowia lipolytica.Appl.Microbiol.Biotechnol.,2016,100,649-659.

[4]Gibson,G.E.；Blass,J.P.；Beal,M.F.；Bunik,V.Theα-ketoglutarate-dehydrogenase complex.Mol.Neurobiol.,2005,31,43-63.

[5]Dumont,M.；Ho,D.J.；Calingasan,N.Y.；Xu,H.；Gibson,G.；Beal,M.F.Mitochondrial dihydrolipoyl succinyltransferase deficiency accelerates amyloid pathology and memory deficit in a transgenic mouse model of amyloid deposition.Free Radic.Biol.Med.,2009,47,1019-1027.

[6]Gibson,G.E.；Starkov,A.；Blass,J.P.；Ratan,R.R.；Beal,M.F.Cause and consequence:mitochondrial dysfunction initiates and propagates neuronal dysfunction,neuronal death and behavioral abnormalities in age-associated neurodegenerative diseases.Biochim.Biophys.Acta,2010,1802,122-134.

[7]Anderson,N.M.；Li,D.；Peng,H.L.；Laroche,F.J.F.；Mansour,M.R.；Gjini,E.；Aioub,M.；Helman,D.J.；Roderick,J.E.；Cheng,T.；Harrold,I.；Samaha,Y.；Meng,L.；Amsterdam,A.；Neuberg,D.S.；Denton,T.T.；Sanda,T.；Kelliher,M.A.；Singh,A.；Look,A.T.；Feng,H.The TCA cycle transferase DLST is important for MYC-mediated leukemogenesis.Leukemia,2016,30,1365-1374.

[8]Heggermont,W.A.；Papageorgiou,A.P.；Quaegebeur,A.；Deckx,S.；Carai,P.；Verhesen,W.；Eelen,G.；Schoors,S.；van Leeuwen,R.；Alekseev,S.；Elzenaar,I.；Vinckier,S.；Pokreisz,P.；Walravens,A.S.；Gijsbers,R.；Haute,C.V.D.；Nickel,A.；Schroen,B.；van Bilsen,M.；Janssens,S.；Maack,C.；Pinto,Y.；Carmeliet,P.；Heymans,S.Inhibition of MicroRNA-146a and Overexpression of Its Target Dihydrolipoyl Succinyltransferase Protect Against Pressure Overload-Induced Cardiac Hypertrophy and Dysfunction.Circulation,2017,136,747–761.

[9]Lycan,T.W.；Pardee,T.S.；Petty,W.J.；Bonomi,M.；Alistar,A.；Lamar,Z.S.；Isom,S.；Chan,M.D.；Miller,A.A.；Ruiz,J.A phase II clinical trial of CPI-613 in patients with relapsed or refractory small cell lung carcinoma.PLoS ONE,2016,11:e0164244.

[10]Pardee,T.S.；Lee,K.；Luddy,J.；Maturo,C.；Rodriguez,R.；Isom,S.；Miller,L.D.；Stadelman,K.M.；Levitan,D.；Hurd,D.；Ellis,L.R.；Harrelson,R.；Manuel,M.；Dralle,S.；Lyerly,S.；Powell,B.L.A phase I study of the first-in-class antimitochondrial metabolism agent,CPI-613,in patients with advanced hematologic malignancies.Clin.Cancer Res.2014,20,5255–5264.

disclosure of Invention

An object of the present invention is to provide a DLST inhibitor.

Another object of the present invention is to provide a composition containing the DLST inhibitor.

It is also an object of the present invention to provide the use of said DLST inhibitors.

It is still another object of the present invention to provide a method for screening DLST inhibitors.

The method provided by the invention is not only suitable for screening the DLST inhibitor under the in vitro condition, but also suitable for screening the DLST inhibitor in living cells.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method of screening for a dihydrolipoic acid succinyltransferase inhibitor, comprising: pretreating an in vitro or living medium with an inhibitor to obtain a pretreatment reaction system; the ex vivo or in vivo medium is selected from a cell lysate, a dihydrolipoic acid succinyltransferase, or a pathogen; preferably, the in vitro or in vivo medium is selected from the group consisting of a cell lysate of rice bacterial leaf blight, a cell lysate of citrus canker pathogen, a recombinant rice bacterial leaf blight dihydrolipoic acid succinyltransferase, rice bacterial leaf blight and citrus canker pathogen.

Preferably, the method further comprises treating the pretreatment reaction system with a probe.

Preferably, the inhibitor is

Preferably, the probe is represented by the following formula I compound:

wherein R is¹Selected from optionally substituted alkylene, imino, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted cycloalkylene, optionally substituted arylene, or optionally substituted heteroarylene; preferably, it is selected from optionally substituted C_1-6Alkylene, optionally substituted C_1-6An arylene group; more preferably, it is selected from methylene, ethylene, propylene, isopropylene, butylene, sec-butylene, pentylene, o-phenylene, m-phenylene, p-phenylene, o-thienyl, m-thienyl, o-furyl, m-furyl, o-pyridyl, m-pyridyl, p-pyridyl, naphthylene, anthrylene, halophenyl; most preferably, it is selected from

R²Selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl; preferably, it is selected from C_1-6Alkyl or C_1-6An aryl group; more preferably, it is selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, pentyl or phenyl.

Most preferably, the probe compound is selected from the following compounds:

1, probe 1: 2- (methylsulfonyl) -5- (4-fluoro-3- (propynyloxy) methyl) phenyl-1, 3, 4-oxadiazole;

and (3) probe 2: 2- (ethylsulfonyl) -5- (4-fluoro-3- (propynyloxy) methyl) phenyl-1, 3, 4-oxadiazole;

and 3, probe 3: 2- (methylsulfonyl) -5- (3- (propynyloxy) methyl) phenyl-1, 3, 4-oxadiazole;

and 4, probe 4: 2- (ethylsulfonyl) -5- (3- (propynyloxy) methyl) phenyl-1, 3, 4-oxadiazole;

and 5, probe: 2- (methylsulfonyl) -5- (2, 4-dichloro-3- (propynyloxy) methyl) phenyl-1, 3, 4-oxadiazole;

and 6, probe 6: 2- (ethylsulfonyl) -5- (2, 4-dichloro-3- (propynyloxy) methyl) phenyl-1, 3, 4-oxadiazole.

Preferably, the system after the probe treatment is Biotin-N₃The chemical reaction is carried out in the presence of a catalyst.

Preferably, the method further comprises the step of performing an imaging reaction analysis.

More preferably, the screening method specifically comprises the following steps:

(1) extracting the total protein of the plant pathogenic bacteria by an ultrasonic crushing method;

(2) incubating the dihydrolipoic acid succinyltransferase inhibitor to be screened and the total protein of the pathogenic bacteria for 2h at 25 ℃, treating blank groups with DMSO, and then respectively treating the total protein for 2h by using a probe capable of labeling the dihydrolipoic acid succinyltransferase;

(3) adding Biotin-N to the above system respectively₃Sodium ascorbate and BTTAA/CuSO₄Mixing the solution, and carrying out click chemistry reaction at 37 ℃;

(4) respectively performing SDS-PAGE on samples, then performing membrane rotation for 50min under an ice bath condition, sealing the samples for 2h by 5% skimmed milk powder, incubating HRP-conjugated Streptavidin at 4 ℃, adding a chemiluminescent substrate, and performing imaging analysis;

or the like, or, alternatively,

(1) carrying out in vitro recombinant expression on the dihydrolipoic acid succinyltransferase;

(2) incubating the dihydrolipoic acid succinyltransferase inhibitor to be screened and the recombinantly expressed dihydrolipoic acid succinyltransferase for 2h at 25 ℃, treating blanks with DMSO, and then adding a probe capable of labeling the dihydrolipoic acid succinyltransferase into the recombinant dihydrolipoic acid succinyltransferase system for labeling;

or the like, or, alternatively,

(1) the dihydrolipoic acid succinyltransferase inhibitors to be screened were incubated with the plant pathogenic bacteria at 25 ℃ for 2h and the blank was treated with DMSO. Then respectively adding probes capable of marking dihydrolipoic acid succinyltransferase to treat the cells for 2 h;

(2) respectively centrifuging the cells, carrying out ultrasonic disruption, and respectively adding Biotin-N into the supernatant₃Sodium ascorbate and BTTAA/CuSO₄Mixing the solution, and carrying out click chemistry reaction at 37 ℃;

(3) after SDS-PAGE is carried out on the samples respectively, the samples are filmed for 50min under the ice bath condition, and are sealed by 5 percent of skimmed milk powder for 2h, and then HRP-conjugated Streptavidin is incubated at 4 ℃, and imaging analysis is carried out after a chemiluminescence substrate is added.

A composition comprising

And pharmaceutically acceptable adjuvants; preferably, the dosage form of the composition is selected from plain tablets, film-coated tablets, sugar-coated tablets, enteric-coated tablets, dispersible tablets, capsules, granules, oral solutions or oral suspensions.

Compound (I)

Can be used for preparing medicines for treating or preventing diseases related to DLST protein; preferably, the diseaseThe disease is Alzheimer disease or leukemia.

Further, the invention also discloses:

a method for screening DLST inhibitors directly in a complex proteome, comprising the steps of:

(2) incubating the DLST inhibitor to be screened and the total protein of the pathogenic bacteria for 2h at 25 ℃, treating blank groups with DMSO, and then respectively treating the total protein for 2h by using a probe 1 or 2 capable of marking DLST;

(4) after SDS-PAGE is carried out on the samples respectively, the samples are filmed for 50min under the ice bath condition, and are sealed by 5 percent of skimmed milk powder for 2h, and then HRP-conjugated Streptavidin is incubated at 4 ℃, and imaging analysis is carried out after a chemiluminescence substrate is added. Inhibitors were screened by comparing the difference in intensity of DLST labeling for probe 1 or 2 in the blank versus the experimental groups. If the labeling strength of the probe 1 or 2 in the experimental group to the DLST is obviously weaker than that in the blank group, the used inhibitor has obvious inhibition activity to the DLST. If there is no significant difference in label intensity, the inhibitor has no significant inhibitory activity on DLST.

A method for screening a DLST inhibitor in vitro by using a recombinant DLST comprises the following steps:

(1) carrying out in vitro recombinant expression on DLST in escherichia coli;

(2) incubating a DLST inhibitor to be screened and DLST of recombinant expression for 2h at 25 ℃, treating a blank group with DMSO, and then adding a probe 1 or 2 capable of labeling the DLST into the system for labeling;

(4) performing SDS-PAGE on the samples respectively, performing membrane rotation for 50min under an ice bath condition, sealing the samples by 5 percent of skimmed milk powder for 2h, incubating HRP-conjugated streptavidin at 4 ℃, adding a chemiluminescent substrate, and performing imaging analysis. Inhibitors were screened by comparing the difference in intensity of DLST labeling for probe 1 or 2 in the blank versus the experimental groups. If the labeling strength of the probe 1 or 2 in the experimental group to the DLST is obviously weaker than that in the blank group, the used inhibitor has obvious inhibition activity to the DLST. If there is no significant difference in label intensity, the inhibitor has no significant inhibitory activity on DLST.

A method for screening DLST inhibitors directly in living cells, which comprises the following steps:

(1) the DLST inhibitors to be screened were incubated with the plant pathogenic bacteria for 2h at 25 ℃ and the blank was treated with DMSO. Then respectively adding a probe 1 or 2 capable of marking DLST to treat the cells for 2 h;

(3) after SDS-PAGE is carried out on the samples respectively, the samples are filmed for 50min under the ice bath condition, and are sealed by 5 percent of skimmed milk powder for 2h, and then HRP-conjugated Streptavidin is incubated at 4 ℃, and imaging analysis is carried out after a chemiluminescence substrate is added. Inhibitors were screened by comparing the difference in intensity of DLST labeling for probe 1 or 2 in the blank versus the experimental groups. If the labeling strength of the probe 1 or 2 in the experimental group to the DLST is obviously weaker than that in the blank group, the used inhibitor has obvious inhibition activity to the DLST. If there is no significant difference in label intensity, the inhibitor has no significant inhibitory activity on DLST.

The invention has the beneficial effects that:

the invention reports a method for screening a DLST inhibitor for the first time, can screen the DLST inhibitor under in vitro and in vivo conditions, and finds that methanesulfonyl bacteria oxazole is an effective DLST inhibitor by the method provided by the invention.

Drawings

FIG. 1; the in vitro screening result of the methylsulfonylconazole on the total protein of the rice bacterial blight (Xoo) is obtained;

FIG. 2; in vitro screening results of methylsulfonylconazole on total protein of citrus canker pathogen (Xac);

FIG. 3; the in vitro screening result of the recombinant expression DLST by the methanesulfonyl bacteria azole;

FIG. 4; the living body screening result of the methylsulfonylconazole on the rice bacterial blight (Xoo);

FIG. 5; the in-vivo screening result of the methylsulfonylconazole on citrus canker pathogen (Xac);

FIG. 6: the invention relates to a flow chart of a screening method.

Examples

The invention is further illustrated by the following examples. It should be understood that the method described in the examples is only for illustrating the present invention and not for limiting the present invention, and that simple modifications of the preparation method of the present invention based on the concept of the present invention are within the scope of the claimed invention. All starting materials and solvents used in the examples are commercially available products of the corresponding purity.

The methylsulfonylconazole has the chemical name of 2- (4-fluorophenyl) -5-methylsulfonyl-1, 3, 4-oxadiazole, is a novel bactericide independently developed by Guizhou university, has the patent number of ZL2011103142462, has broad-spectrum and efficient antibacterial activity on various plant pathogenic bacteria, and has a good development prospect. The structural formula is as follows:

example 1: in vitro screening result of methylsulfonylconazole on total protein of rice bacterial blight (Xoo)

100mL of OD was taken₆₀₀Xoo broth (0.6), was centrifuged at 7500rpm for 10min, the supernatant was discarded, and then washed 3 times with PBS. The cells were resuspended in 10mL of PBS (pH 7.2), the centrifuge tube was placed on ice for ultrasonication, and the protein concentration was measured by bradford method, followed by dilution to 1 mg/mL. mu.L of total protein was taken, 1. mu.L of methanesulfonylazole was added thereto at various concentrations to give final concentrations of 50, 100, 250, 500. mu.M, and an equal volume of DMSO was added to the blank group, and the mixture was incubated at 25 ℃ for 2 hours, respectively. Then 5. mu.M of Probe 1 was added, and incubation was continued for 2 h. Adding biotin-N into the above system respectively₃Ascorbic acidSodium and BTTAA/CuSO₄The mixed solution was subjected to click reaction. After 2h, 2 × loading buffer was added to the system and boiled at 95 deg.C for 10 min. 10 mu g of each sample was subjected to SDS-PAGE, then subjected to membrane rotation for 50min at a constant current of 200mA, and washed with PBST and then blocked with 5% skimmed milk powder for 2 h. Washing the membrane with PBST, adding HRP-labeled Streptavidin (HRP-Streptavidin) for incubation for 2h, then adding HRP-labeled Streptavidin (HRP-Streptavidin) for incubation for 2h, washing the membrane with PBST, adding a chemiluminescent reagent, and finally performing imaging analysis by a chemiluminescent imaging system (as shown in FIG. 1). As can be seen from fig. 1, compared with the blank control group, the labeling intensity of probe 1 on DLST gradually decreases with the increase of the concentration of methanesulfonyl bacteria azole in the experimental group, which indicates that methanesulfonyl bacteria azole can effectively inhibit DLST activity under the in vitro condition.

Example 2: in vitro screening result of methylsulfonylconazole on total protein of citrus canker pathogen (Xac)

100mL of OD was taken₆₀₀The cells were centrifuged at 7500rpm for 10min under 0.6 Xac (Amersham biosciences) and the supernatant was discarded, followed by 3-time washing with PBS. The cells were resuspended in 10mL of PBS (pH 7.2), the centrifuge tube was placed on ice for ultrasonication, and the protein concentration was measured by bradford method, followed by dilution to 1 mg/mL. mu.L of total protein was taken, 1. mu.L of methanesulfonylazole was added thereto at various concentrations to give final concentrations of 10, 25, 50, 100, 250. mu.M, and an equal volume of DMSO was added to the blank group, and the mixture was incubated at 25 ℃ for 2 hours, respectively. Then 50. mu.M probe 2 was added separately and incubation continued for 2 h. Adding biotin-N into the above system respectively₃Sodium ascorbate and BTTAA/CuSO₄The mixed solution undergoes a click chemistry reaction. After 2h, 2 × loading buffer was added to the system and boiled at 95 deg.C for 10 min. 10 μ g of each sample was subjected to SDS-PAGE, followed by membrane rotation at 200mA for 50min, PBST membrane washing and blocking with 5% skimmed milk powder for 2 h. Washing the membrane with PBST, adding HRP-labeled Streptavidin (HRP-Streptavidin) for incubation for 2h, then adding HRP-labeled Streptavidin (HRP-Streptavidin) for incubation for 2h, washing the membrane with PBST, adding a chemiluminescent reagent, and finally performing imaging analysis by a chemiluminescent imaging system (as shown in FIG. 2). As can be seen from FIG. 2, the test group showed an increase in the concentration of methanesulfonylazole as compared with the blank control groupThe labeling intensity of the probe 2 on DLST is gradually weakened, which shows that the methanesulfonylazole can effectively inhibit the activity of DLST in Xac under the condition of vitro.

Example 3: in vitro screening result of mesylatazole on recombinant expression DLST

And (3) constructing a recombinant plasmid by taking pet28a as a vector, introducing the recombinant plasmid into a BL21 strain, carrying out amplification culture, carrying out affinity purification on the recombinant plasmid through a Ni column, carrying out enzyme digestion through thrombin, and finally purifying through a molecular sieve to obtain the recombinant DLST. 25ng of recombinant DLST was taken and biotin-N was added₃Sodium ascorbate and BTTAA/CuSO₄The mixed solution undergoes a click chemistry reaction. Then adding 2 × loading buffer into the system respectively and boiling for 10min at 95 ℃. 12.5ng of the recombinant DLST was subjected to SDS-PAGE, and then membrane-transferred at 200mA for 50min, and washed with PBST and then blocked with 5% skimmed milk powder for 2 h. Washing the membrane with PBST, adding HRP-labeled Streptavidin (HRP-Streptavidin) for incubation for 2h, then adding HRP-labeled Streptavidin (HRP-Streptavidin) for incubation for 2h, washing the membrane with PBST, adding a chemiluminescent reagent, and finally performing imaging analysis by a chemiluminescent imaging system (as shown in FIG. 3). As can be seen from FIG. 3, in the experimental group, the labeling intensity of probe 2 on the recombinant DLST is gradually reduced with the increase of the concentration of methanesulfonyl bacteria azole, compared with the blank control group, which indicates that methanesulfonyl bacteria azole can effectively inhibit the activity of recombinant DLST.

Example 4: results of in vivo screening of rice bacterial blight disease (Xoo) by methanesulfonylazole

Respectively taking 2mL of OD₆₀₀The supernatant was discarded after centrifugation of 0.6 Xoo, the cells were resuspended in PBS (pH 7.2), 25, 50, 100, 250, 500. mu.M methanesulfonylazole was added to the system for 2h, and the blank was treated with DMSO. Then 5. mu.M of Probe 1 was added, and incubation was continued for 2 h. Centrifuging, discarding supernatant, washing for 3 times by PBS, adding 100 μ L PBS to resuspend cells, ultrasonically crushing cells, centrifuging at 12000rpm for 30min, measuring protein concentration of supernatant by bradford method, and diluting to 1 μ g/μ L. Respectively taking 45 mu L of supernatant, and respectively adding biotin-N₃Sodium ascorbate and BTTAA/CuSO₄The mixed solution is subjected to click chemistry reaction, and an equal volume of 2 × loading buffer is added into the sample respectively, and the sample is boiled for 10min at 95 ℃. Is divided intoSamples of 10. mu.g each were subjected to SDS-PAGE, then subjected to membrane rotation at 200mA for 50min, washed with PBST and then blocked with 5% skimmed milk powder for 2 h. Washing the membrane with PBST, adding HRP-labeled Streptavidin (HRP-Streptavidin) for incubation for 2h, then adding HRP-labeled Streptavidin (HRP-Streptavidin) for incubation for 2h, washing the membrane with PBST, adding a chemiluminescent reagent, and finally performing imaging analysis by a chemiluminescent imaging system (as shown in FIG. 4). As can be seen from FIG. 4, the labeling intensity of probe 1 on DLST gradually decreased with the increase of the concentration of methanesulfonyl bacteria azole in the experimental group compared to the blank control group, indicating that methanesulfonyl bacteria azole can effectively inhibit DLST activity in Xoo cells.

Example 5: in-vivo screening result of methanesulfonyl bacteria azole on citrus canker pathogen (Xac)

Respectively taking 2mL of OD₆₀₀The cells were resuspended in 0.98mL PBS (pH 7.2), 10, 25, 50, 100, 250 μ M methanesulfonyl azole was added to the system for 2h, and the blank was treated with DMSO. Then 5. mu.M probe 2 was added separately and incubation continued for 2 h. Centrifuging, discarding supernatant, washing for 3 times by PBS, adding 100 μ L PBS to resuspend cells, ultrasonically crushing cells, centrifuging at 12000rpm for 30min, measuring protein concentration of supernatant by bradford method, and diluting to 1 μ g/μ L. Respectively taking 45 mu L of supernatant, and respectively adding biotin-N₃Sodium ascorbate and BTTAA/CuSO₄The mixed solution is subjected to click chemistry reaction, and an equal volume of 2 × loading buffer is added into the sample respectively, and the sample is boiled for 10min at 95 ℃.10 μ g of each sample was subjected to SDS-PAGE, followed by membrane rotation at 200mA for 50min, PBST membrane washing and blocking with 5% skimmed milk powder for 2 h. Then adding HRP labeled Streptavidin (HRP-Streptavidin) for incubation for 2h, washing the membrane by PBST, adding a chemiluminescent reagent, and finally performing imaging analysis by a chemiluminescent imaging system (as shown in FIG. 5). As can be seen from FIG. 5, the labeling intensity of probe 2 on DLST gradually decreased with the increase of the concentration of methanesulfonyl azole in the experimental group compared to the blank control group, indicating that methanesulfonyl azole can effectively inhibit DLST activity in Xac cells.

Example 6: preparation of probes 1 to 6

(1) 0.20mol of the variously substituted benzoic acid was taken in a 250mL round-bottom flask, 20-fold equivalent of methanol and 4mL of 98% concentrated sulfuric acid were added thereto, and then the reaction was refluxed for 4 to 6 hours. Removing methanol by reduced pressure distillation, mixing with silica gel, and purifying by column chromatography to obtain compound 1 with yield of 96-99%.

(2) 0.15mol of Compound 1 was placed in a 100mL round-bottom flask, and 2.5-fold equivalent of 80% hydrazine hydrate was added thereto and reacted under reflux for 2 hours. After the system is cooled, white solid is separated out, and the compound 2 is obtained by washing and drying, wherein the yield is 77-91%.

(3) Taking 0.15mol of compound 2 in a 250mL round-bottom flask, adding 2 times of equivalent of KOH and 100mL of ethanol, stirring to dissolve, and then dropwise adding 2 times of equivalent of CS₂Stirring at normal temperature for 8h, and then carrying out reflux reaction for 24-36 h. Distilling under reduced pressure to remove ethanol, adjusting pH to 4-5 with 5% diluted hydrochloric acid in ice bath, and vacuum filtering to obtain crude product 3.

(4) 0.10mol of the crude product 3 was taken in a 100mL round-bottom flask, 50mL of an aqueous solution containing 0.20mol of KOH was added thereto, and 2-fold equivalent of dimethyl (ethyl) sulfate was slowly added dropwise thereto and stirred at room temperature for 2 hours. And (3) carrying out suction filtration on the system to obtain a yellow solid, dissolving a small amount of ethyl acetate, mixing the sample with silica gel, and carrying out column chromatography purification to obtain a compound 4, wherein the yield of the two steps is 36-53%.

(5) 0.03mol of Compound 4 was taken in a 100mL round-bottom flask, and 50mL of dry CCl was added thereto₄Then, 1.2 equivalent of NBS and 0.1 equivalent of AIBN were added and the reaction was refluxed for 48 hours. Filtering the reaction system to remove insoluble substances, and then distilling under reduced pressure to remove CCl₄Taking a small amount of CH₂Cl₂Dissolving the solid, mixing the solid with silica gel, and purifying by column chromatography to obtain compound 5 with yield of 43-77%.

(6) 5mmol of compound 5 and 1.1-fold equivalent of propargyl alcohol were taken in a 100mL round-bottomed flask, 20mL of anhydrous DMF was added thereto, dissolved with stirring in ice bath, and then 1.2-fold equivalent of NaH was added thereto for 2 h. Adding saturated 20mL of ammonium chloride solution and 60mL of ethyl acetate into the system for extraction, extracting the water phase with 50mL of ethyl acetate for 2 times, combining the organic phases, removing the solvent by reduced pressure distillation, mixing the sample with silica gel, and purifying by column chromatography to obtain the compound 6 with the yield of 43-66%.

(7) 5mmol of Compound 6 dissolved in 15mL of dry CH₂Cl₂Stirring in ice bathThe mixture is stirred and dissolved, and then 3 times of equivalent of mCPBA is added into the system for reaction for 2 hours. Filtering the reaction system to remove solid insoluble substances, and distilling under reduced pressure to remove part of CH₂Cl₂And mixing the rest system with silica gel, and purifying by column chromatography to obtain target product probe 1-6 with yield of 40-70%.

The reaction scheme is as follows:

the structure of probes 1-6 is characterized as follows:

1, probe 1: 2- (methylsulfonyl) -5- (4-fluoro-3- (propynyloxy) methyl) phenyl-1, 3, 4-oxadiazole;¹H NMR(500MHz,CDCl₃)δ8.26(dd,J＝6.6,2.2Hz,1H),8.10(ddd,J＝8.5,4.8,2.3Hz,1H),7.25(t,J＝8.9Hz,1H),4.74(s,2H),4.30(d,J＝2.4Hz,2H),3.53(s,3H),2.52(t,J＝2.4Hz,1H).¹³C NMR(125MHz,CDCl₃)δ165.89,164.48,162.43,162.23,129.89,129.57,127.10,118.53,116.95,78.98,75.54,64.53,58.24,43.08.HRMS(ESI)calculated for C₁₃H₁₁FN₂O₄S[M+H]⁺m/z 311.0496,found 311.0493.

and (3) probe 2: 2- (ethylsulfonyl) -5- (4-fluoro-3- (propynyloxy) methyl) phenyl-1, 3, 4-oxadiazole;¹H NMR(500MHz,CDCl₃)δ8.26(dd,J＝6.6,2.3Hz,1H),8.10(ddd,J＝8.5,4.8,2.3Hz,1H),7.25(t,J＝8.9Hz,1H),4.75(s,2H),4.31(d,J＝2.4Hz,2H),3.62(q,J＝7.4Hz,2H),2.52(t,J＝2.4Hz,1H),1.55(t,J＝7.4Hz,3H).¹³C NMR(125MHz,CDCl₃)δ165.90,164.46,162.42,161.42,129.90,129.56,127.08,118.59,116.92,78.98,75.50,64.50,58.23,50.16,6.92.HRMS(ESI)calculated for C₁₄H₁₃FN₂O₄S[M+H]⁺m/z 325.0653,found325.0648.

and 3, probe 3: 2- (methylsulfonyl) -5- (3- (propynyloxy) methyl) phenyl-1, 3, 4-oxadiazole;¹H NMR(500MHz,CDCl₃)δ8.12(d,J＝8.4Hz,2H),7.55(d,J＝8.5Hz,2H),4.70(s,2H),4.26(d,J＝2.4Hz,2H),3.53(s,3H),2.51(t,J＝2.4Hz,1H).¹³C NMR(125MHz,CDCl₃)δ166.57,162.17,143.50,128.49,127.96,121.44,79.21,75.28,70.78,57.88,43.08.HRMS(ESI)calculated for C₁₃H₁₂N₂O₄S[M+H]⁺m/z 293.0591,found 293.0587.

and 4, probe 4: 2- (ethylsulfonyl) -5- (3- (propynyloxy) methyl) phenyl-1, 3, 4-oxadiazole;¹H NMR(500MHz,CDCl₃)δ8.11(d,J＝8.3Hz,2H),7.55(d,J＝8.1Hz,2H),4.70(s,2H),4.25(d,J＝2.3Hz,2H),3.61(q,J＝7.4Hz,2H),2.50(t,J＝2.4Hz,1H),1.54(t,J＝7.4Hz,3H).¹³C NMR(125MHz,CDCl₃)δ166.59,161.37,143.47,128.47,127.94,121.53,79.22,75.25,70.79,57.87,50.17,6.91.HRMS(ESI)calculated for C₁₄H₁₄N₂O₄S[M+H]⁺m/z 307.0747,found307.0744.

and 5, probe: 2- (methylsulfonyl) -5- (2, 4-dichloro-3- (propynyloxy) methyl) phenyl-1, 3, 4-oxadiazole;¹H NMR(500MHz,CDCl₃)δ7.93(d,J＝8.5Hz,1H),7.54(d,J＝8.5Hz,1H),4.97(s,2H),4.30(d,J＝2.4Hz,2H),3.54(s,3H),2.52(t,J＝2.4Hz,1H).¹³C NMR(125MHz,CDCl₃)δ164.55,162.69,141.97,136.88,135.66,132.18,129.01,121.33,79.12,75.33,66.34,58.53,43.07.HRMS(ESI)calculated for C₁₃H₁₀N₂O₄SCl₂[M+H]⁺m/z 360.9811,found360.9807.

and 6, probe 6: 2- (ethylsulfonyl) -5- (2, 4-dichloro-3- (propynyloxy) methyl) phenyl-1, 3, 4-oxadiazole;¹H NMR(500MHz,CDCl₃)δ7.93(d,J＝8.5Hz,1H),7.54(d,J＝8.5Hz,1H),4.97(s,2H),4.30(d,J＝2.4Hz,2H),3.62(q,J＝7.5Hz,2H),2.52(t,J＝2.4Hz,1H),1.56(t,J＝7.4Hz,3H).¹³C NMR(125MHz,CDCl₃)δ164.60,161.94,141.92,136.84,135.64,132.18,129.00,121.42,79.12,75.30,66.33,58.51,50.25,6.94.HRMS(ESI)calculated for C₁₄H₁₂N₂O₄SCl₂[M+H]⁺m/z 374.9968,found 374.9966.

the above description is only a preferred embodiment of the present invention, and the implementation manner of the present invention is not limited by the above embodiment. Modifications may be made by those skilled in the art without departing from the principles of the invention and are intended to be included within the scope of the invention.

Claims

1. A screening method of a dihydrolipoic acid succinyltransferase inhibitor, characterized by comprising:

pretreating an in vitro or living medium by using an inhibitor to obtain a pretreatment reaction system; the ex vivo or in vivo medium is selected from a cell lysate, a dihydrolipoic acid succinyltransferase, or a pathogen;

then treating the pretreatment reaction system by using a probe;

the inhibitor is

The probe is shown as the following compound of formula I:

wherein R is¹Selected from optionally substituted alkylene, imino, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted cycloalkylene, optionally substituted arylene, or optionally substituted heteroarylene;

R²selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl;

the system after the probe treatment is Biotin-N₃Carrying out click chemistry reaction in the presence of the catalyst;

then, the imaging reaction analysis is carried out.

2. The screening method according to claim 1, wherein: the cell lysate, the dihydrolipoic acid succinyltransferase or the pathogen is selected from the group consisting of a rice bacterial leaf blight germ cell lysate, a citrus canker germ cell lysate, a recombinant rice bacterial leaf blight germ dihydrolipoic acid succinyltransferase, rice bacterial leaf blight germ and citrus canker germ.

3. The screening method according to claim 1, wherein: r¹Selected from optionally substituted C_1-6Alkylene, optionally substituted C_1-6An arylene group.

4. The screening method according to claim 1, wherein: r¹Selected from the group consisting of methylene, ethylene, propylene, isopropylene, butylene, sec-butylene, pentylene, o-phenylene, m-phenylene, p-phenylene, o-thienyl, m-thienyl, o-furyl, m-furyl, o-pyridyl, m-pyridyl, p-pyridyl, naphthylene, anthracenylene, halophenyl.

5. The screening method according to claim 1, wherein: r¹Is selected from

6. The screening method according to claim 1, wherein: r²Is selected from C_1-6Alkyl or C_1-6And (4) an aryl group.

7. The screening method according to claim 1, wherein: r²Selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, pentyl or phenyl.

8. The screening method according to claim 1, wherein: the probe compound is selected from the following compounds:

9. The screening method according to claim 1, characterized in that it comprises in particular the following steps:

(4) after SDS-PAGE is carried out on the samples respectively, the samples are filmed for 50min under the ice bath condition, and are sealed by 5 percent of skimmed milk powder for 2h, and then HRP-conjugated Streptavidin is incubated at 4 ℃, and imaging analysis is carried out after a chemiluminescence substrate is added.

10. The screening method according to claim 1, characterized in that it comprises in particular the following steps:

(1) carrying out in-vitro recombinant expression on dihydrolipoic acid succinyltransferase in rice bacterial leaf blight bacteria;

(3) adding Biotin-N to the above system respectively₃Sodium ascorbateAnd BTTAA/CuSO₄Mixing the solution, and carrying out click chemistry reaction at 37 ℃;

(4) performing SDS-PAGE on the samples respectively, performing membrane rotation for 50min under an ice bath condition, sealing the samples by 5 percent of skimmed milk powder for 2h, incubating HRP-conjugated streptavidin at 4 ℃, adding a chemiluminescent substrate, and performing imaging analysis.

11. The screening method according to claim 1, characterized in that it comprises in particular the following steps:

(1) incubating dihydrolipoic acid succinyltransferase inhibitors to be screened with plant pathogenic bacteria at 25 ℃ for 2h, treating the blank with DMSO; then respectively adding probes capable of marking dihydrolipoic acid succinyltransferase to treat the cells for 2 h;

12. A pharmaceutical composition for treating or preventing diseases associated with dihydrolipoic acid succinyltransferase protein, characterized by comprising

And pharmaceutically acceptable adjuvants; the dosage form of the composition is selected from plain tablets, film-coated tablets, sugar-coated tablets, enteric-coated tablets, dispersible tablets, capsules, granules, oral solution or oral suspension.

13. Compound (I)

In the preparation of medicaments for the treatment or prophylaxis of diseases which are associated with dihydrolipoate succinyltransferase proteinsThe application in the aspect of medicines.

14. The use according to claim 13, wherein the disease is alzheimer's disease, leukemia or cardiovascular disease.