阅读说明：本技术 一种细菌核糖体抑制剂筛选方法及其抑制剂 (Bacterial ribosome inhibitor screening method and inhibitor thereof ) 是由 罗有福 杨涛 桑梓苔 于 2019-04-12 设计创作，主要内容包括：本发明涉及一种细菌核糖体抑制剂筛选方法及其抑制剂,其特征在于该筛选体系为细菌核糖体粗提物介导的原位点击化学反应偶联细菌体外转录/翻译体系。向细菌核糖体粗提物中分别加入合成砌块,而后加入荧光素酶质粒和细菌转录/翻译体系反应试剂,最后利用荧光素酶检测系统和酶标仪测定体系荧光强度值,下降越多者对应的原位点击化学产物极为活性越好的细菌核糖体小分子抑制剂。本发明同时公开了利用上述筛选方法得到的一系列新型细菌核糖体抑制剂及其在制备抗金黄色葡萄球菌药物方面的用途。本发明公开的细菌核糖体抑制剂筛选方法及其抑制剂为开发和制备抗菌药物方面具有极好的应用前景。(The invention relates to a screening method of a bacterial ribosome inhibitor and the inhibitor thereof, which is characterized in that the screening system is an in-situ click chemistry reaction coupling bacterial in-vitro transcription/translation system mediated by a bacterial ribosome crude extract. And (2) respectively adding the synthetic building blocks into the crude extract of the bacterial ribosome, then adding a luciferase plasmid and a bacterial transcription/translation system reaction reagent, and finally determining the fluorescence intensity value of the system by using a luciferase detection system and a microplate reader, wherein the more the fluorescence intensity value is reduced, the more the corresponding in-situ click chemical product is reduced, and the bacterial ribosome small-molecule inhibitor with better activity is obtained. The invention also discloses a series of novel bacterial ribosome inhibitors obtained by the screening method and application thereof in preparing anti-staphylococcus aureus medicaments. The screening method of the bacterial ribosome inhibitor and the inhibitor thereof disclosed by the invention have excellent application prospects in the aspects of developing and preparing antibacterial drugs.)

1. A bacterial ribosome inhibitor screening method is characterized by being a high-throughput screening system for chemical synthesis coupling activity test.

2. The method of claim 1, wherein the chemical synthesis stage is an in situ click chemistry reaction.

3. An in situ click chemistry reaction as claimed in claim 2 wherein said biocatalytic template is a crude bacterial ribosome extract.

4. An in situ click chemistry reaction as claimed in claim 2 wherein the reaction building blocks are azido building blocks and alkyne-containing building blocks.

5. An in situ click chemistry reaction as claimed in claim 2 wherein the reaction type is a [2+3] dipolar cycloaddition reaction.

6. The crude bacterial ribosome extract of biological templates for catalyzing click chemistry reactions in situ according to claim 3, characterized by the steps of: firstly, culturing bacteria in a liquid culture medium at 37 ℃ until the OD is 600-2, then carrying out high-speed centrifugation (4000rpm for 20 minutes) at low temperature (4 ℃) to collect thalli, crushing the thalli by a high-pressure homogenizer and a cell crusher, and finally carrying out low-temperature high-speed centrifugation (12,000RCF, SS-34,10,000rpm,10 minutes, 4 ℃) for 10 minutes to obtain a bacterial ribosome crude extract.

7. A biocatalytic template for in situ click chemistry reactions as in claim 3, wherein the biocatalytic template is derived from Escherichia coli or Staphylococcus aureus.

8. A bacterial ribosome screening method according to claim 1, in which the activity test phase is a bacterial coupled transcription/translation system.

9. The method for screening a bacterial ribosome inhibitor according to claim 1, which comprises the following steps: 1) preparing a click chemistry biological catalysis template: the bacteria were cultured to OD in bacterial culture medium in a shaker at 37 ℃₆₀₀After 2, collecting thalli by low-temperature high-speed centrifugation, crushing the thalli by a high-pressure homogenizer and a cell crusher, and finally obtaining a bacterial ribosome crude extract by 10min low-temperature high-speed centrifugation (12,000RCF, SS-34,10,000rpm,10min,4 ℃); 2) incubating the crude bacterial ribosome extract with azide building blocks (5 μ M) at 0 ℃ for 30min, adding alkynyl building blocks (5mM), incubating at room temperature for 24h, wherein in-situ click chemistry reaction occurs to generate corresponding products; 3) the system was supplemented with a mixture of materials occurring in the bacterial in vitro transcription/translation system [500mM potassium acetate, 87.5mM Tris-acetate [ pH8.0]]67.5mM ammonium acetate, 50g/mL of folinic acid, 5mM DTT,87.5mg/mL polyethylene glycol, 5.0mM ATP,1.25mM [ each]Ribonucleoside triphosphate, 50mM c,2.5mM cyclic AMP,250g/mL of E.coli tRNA]Amino acid mixture (1.25 mM each amino acid concentration), fluorescent plasmid pSAluc]Co-incubation at 37 ℃ for 1 hour; 4) and (3) measuring the fluorescence intensity value of the system by using a luciferase detection system and a microplate reader.

10. The bacterial ribosome inhibitor according to claim 1, which is characterized by comprising a small molecule compound 2c and a class of small molecule compounds represented by the general structural formula (I):

in the general structural formula (I), R1 is hydrogen element, or mono-substituted or multi-substituted F, Cl, Br, nitro, methoxy, acetyl and trifluoromethoxy at 1,2,3, 4 position; r₂Is hydrogen element or monosubstituted or polysubstituted F at the positions 5, 6, 7, 8 and 9.

11. A series of bacterial ribosome inhibitors according to claim 10, characterised by being selected from the following compounds:

2- (1- (2,4', 6-trifluoro-ether- [1,1' -biphenyl ] -4-yl) -1H-1,2, 3-triazole-4-yl) pyridine (2a)

1- (3'- (4- (2-pyridyl) -1H-1,2, 3-triazole-1-yl) - [1,1' -biphenyl ] -4-yl) ethanone (2b)

2- (1- (4- ([1,3] dioxol-5-yl) -2-fluorophenyl) -1H-1,2, 3-triazol-4-yl) pyridine (2c)

2- (1- (2,3', 6-trifluoro-ether- [1,1' -biphenyl ] -4-yl) -1H-1,2, 3-triazole-4-yl) pyridine (2d)

1- (4'- (4- (pyridin-2-yl) -1H-1,2, 3-triazol-1-yl) - [1,1' -biphenyl ] -4-yl) ethanone (2e)

2- (1- (3, 5-difluoro-3 ',4' -dimethoxy- [1,1' -biphenyl ] -4-yl) -1H-1,2, 3-triazole-4-yl) pyridine (2f)

2- (1- (2,2', 6-trifluoro-1, 1' -biphenyl ] -4-yl) -1H-1,2, 3-triazol-4-yl) pyridine (2g)

2- (1- (3'- (trifluoromethoxy) - [1,1' -biphenyl ] -4-yl) -1H-1,2, 3-triazole-4-yl) pyridine (2H)

2- (1- ([1,1' -biphenyl ] -4-yl) -1H-1,2, 3-triazol-4-yl) pyridine (2i)

Technical Field

The invention relates to the field of antibacterial drug research, in particular to a series of bacterial ribosome inhibitors and a method for screening the bacterial ribosome inhibitors of a novel in-situ click chemistry coupling bacterial in-vitro translation system constructed by utilizing a bacterial ribosome crude extract.

Background

Click chemistry (Click chemistry), also known as link chemistry, dynamic combinatorial chemistry, and the like. The method is a new combined chemistry method which rapidly completes the chemical synthesis of various molecules through the splicing of small units and establishes the basis of the carbon heteroatom bond (C-X-C) synthesis. Click chemistry reactions generally have the following characteristics: the reaction raw materials are easy to obtain; the reaction operation is simple, and the conditions are mild; the product yield is high, and the stereoselectivity of the reaction is good; the reaction is rapid, and the dynamic driving force is high; the post-treatment process of the obtained product is simple; according with atom economy and the like. Click chemistry minimizes the adverse environmental impact of chemical reactions. The click chemistry reactions currently applied in drug development mainly involve the following 4 reaction types: cycloaddition reactions, affinity ring-opening reactions, non-aldol carbonyl chemistry, and carbon-carbon multiple bond addition reactions.

In situ click chemistry (In situ click chemistry) is currently the most widely used method for click chemistry In synthetic chemistry. In the synthesis process, substances which have click reaction are generated in the solution, and the click reaction reagent is directly added into the reaction system without the processing steps of separation, purification and the like, and the reaction is started. For example, an enzyme is used as an in situ click chemistry template to link the module components to synthesize an inhibitor of the enzyme itself. This approach has been successfully applied to a number of examples of the discovery of inhibitor molecules for drug target proteins. Including acetylcholinesterase, bacterial ribosomes, and the like.

The traditional in-situ click chemistry method using bacterial ribosome as a biological template comprises the following steps: firstly, bacteria such as escherichia coli are cultured, and then the bacteria ribosome is obtained through more complicated separation and purification steps such as bacteria collection, bacteria breaking, sucrose gradient centrifugation and ultracentrifugation. The click chemistry reaction module and the separated and purified bacterial ribosome are sequentially incubated in a suitable solution at a moderate temperature for a certain period of time to enable click chemistry reaction to occur, such as azide-alkynyl cycloaddition reaction and the like. After the reaction is finished, analyzing the in-situ click chemical reaction sample by using a liquid chromatography-mass spectrometry (LC-MS) analysis method to detect a click chemical reaction final product. Related documents report that the ribosome inhibitor small molecule compound based on the solithromycin framework with antibacterial activity is screened and found by using the method.

Among the prior in situ click chemistry product analysis methods, the most common one is LC-MS analysis. Compared with the bacterial in-vitro translation system in the coupling screening platform, the LC-MS method for detecting reaction products is difficult to realize high-throughput screening, and is particularly shown in the following steps: 1) the click chemical reaction system still needs pretreatment processes such as sample precipitation to remove biological template protein and the like to carry out LC-MS sample injection detection; 2) the liquid phase separation and mass spectrum conditions of LC-MS need to be debugged in advance to achieve the best detection effect 3) LC-MS analysis samples need to be transferred into a sample injection bottle and are sequentially analyzed one by one, and even at the analysis speed of 5 minutes/sample, higher-flux screening cannot be achieved.

In the invention, an in-situ click chemistry process is organically coupled with a bacteria in-vitro transcription/translation system. The bacterial in vitro translation system is a process for synthesizing the required protein by simulating bacteria in a non-cell system in vitro through transcription and translation processes under the coordination of ribosome and other necessary materials. The following steps are specified for the usual study examples: firstly, constructing a plasmid which contains a fluorescent protein gene and can be transcribed and translated in a bacterial system; subsequently, the plasmid is mixed with all materials required for the transcription and translation of the bacteria, such as amino acid mixture, ATP, CTP and GTP, etc., typically in a microplate or PCR tube, and the system is incubated at a suitable temperature to allow the plasmid to be transcribed and translated in vitro to produce the fluorescent protein. The change of the expression level of the protein reflects the change of the ribosome bioactivity. The invention utilizes the biological activity of a biological target, namely bacterial ribosome to carry out high-throughput screening on a small molecule product obtained by in-situ click chemical reaction so as to obtain the effective bacterial ribosome small molecule inhibitor.

For the determination of the amount of the fluorescent protein finally produced by the transcription and translation process involving the ribosome, the invention uses a luminescent Kinase assay system, and a large number of commercial kits are available for this step (for example, Kinase-Luminescense kinase assay platform Kit, etc.). The principle of the kit is that in the presence of ATP, a test substrate fluorescein (provided in the kit) generates oxidized fluorescein under the enzyme catalysis of fluorescent protein and simultaneously emits fluorescence, and an enzyme-labeling instrument is used for detecting the fluorescence intensity so as to measure the content of catalytic enzyme, namely the fluorescent protein.

Disclosure of Invention

The invention aims to provide a bacterial ribosome inhibitor screening method and a bacterial ribosome inhibitor, which overcome the defects of the existing method.

A method of screening for bacterial ribosome inhibitors as claimed in claim 1 wherein: is a high-throughput screening system for chemical synthesis coupling activity test.

The method of claim 1, wherein the chemical synthesis stage is an in situ click chemistry reaction.

The biocatalytic template for in situ click chemistry reaction of claim 2 is crude bacterial ribosome extract.

An in situ click chemistry reaction as claimed in claim 2 wherein the reaction building blocks are azido building blocks and alkyne-containing building blocks.

An in situ click chemistry reaction as claimed in claim 2 wherein the reaction type is a [2+3] dipolar cycloaddition reaction.

The crude extract of bacterial ribosome of the in-situ click chemistry biological template of the invention as claimed in claim 3 is prepared by the following steps: culturing bacteria to a certain stage, centrifuging, collecting bacteria, and breaking bacteria. The method comprises the following specific steps: culturing the bacteria in a shaking table at 37 ℃ to OD 600-2 in a bacteria culture medium, and then centrifuging at a low temperature and a high speed to collect the bacteria. The thalli are crushed by a high-pressure homogenizer and a cell crusher. Finally, the crude extract of bacterial ribosome was obtained by 10min low temperature high speed centrifugation (12,000RCF, SS-34,10,000rpm,10min,4 ℃).

The crude bacterial ribosome extract of claim 3 can be derived from E.coli or S.aureus.

The in situ click chemistry synthesis building blocks of the invention as described in claim 2 are azido building blocks and alkyne-containing building blocks.

The method for screening a bacterial ribosome inhibitor according to claim 1, wherein the process of generating protein by ribosome translation based on biological target activity is coupled with in-situ click chemistry reaction, and triazole cycloaddition products generated by crude ribosome extract as in-situ click chemistry biocatalysis template are subjected to high-throughput screening to obtain the effective ribosome small-molecule inhibitor.

The method for screening a bacterial ribosome inhibitor according to claim 1, comprising the following steps: in a multi-well plate, the crude bacterial ribosome extract was incubated with the alkynyl building block (5 μ M) for 30min at 0 ℃ followed by the addition of the azido building block (50 μ M) and incubation at room temperature for 24h, during which in situ click chemistry reactions occurred to generate the corresponding product. Subsequently, a mixture of materials occurring in the bacterial in vitro transcription/translation system [500mM potassium acetate, 87.5mM Tris-acetate [ pH8.0],67.5mM amine acetate, 50g/mL of folinic acid, 5mM DTT,87.5mg/mL polyethylene glycol, 5.0mM ATP,1.25mM [ each ] ribonucleoside triphosphate, 50mM c,2.5mM cyclic AMP,250g/mL of Escherichia coli tRNA ], an amino acid mixture (each amino acid concentration being 1.25mM), and a fluorescent plasmid pSAluc ] were added and incubated together at 37 ℃ for 1 hour, during which the ribosomes in the crude extract of bacterial ribosomes exert their biological activities of transcribing and translating proteins to produce fluorescent proteins, and the fluorescent proteins were quantified by the luminescence kinase assay system to measure the biological activities of the bacterial ribosomes and the extent to which they are inhibited.

A series of bacterial ribosome inhibitors as claimed in claim 1, comprising a small molecule compound of chemical structure 2c and a class of small molecule compounds of general structural formula (I):

in the general structural formula (I), R1 is hydrogen element, or mono-substituted or multi-substituted F, Cl, Br, nitro, methoxy, acetyl and trifluoromethoxy at 1,2,3, 4 position; r₂Is hydrogen element or monosubstituted or polysubstituted F at the positions 5, 6, 7, 8 and 9.

The small molecule compound 2c is: 3- (1- (4- ([1,3] dioxol-5-yl) -2-fluorophenyl) -1H-1,2, 3-triazol-4-yl) pyridine (2c)

2- (1- (2,4', 6-trifluoro-ether- [1,1' -biphenyl ] -4-yl) -1H-1,2, 3-triazole-4-yl) pyridine (2a)

1- (3'- (4- (2-pyridyl) -1H-1,2, 3-triazole-1-yl) - [1,1' -biphenyl ] -4-yl) ethanone (2b)

3- (1- (2,3', 6-trifluoro-ether- [1,1' -biphenyl ] -4-yl) -1H-1,2, 3-triazole-4-yl) pyridine (2d)

1- (4'- (4- (pyridin-2-yl) -1H-1,2, 3-triazol-1-yl) - [1,1' -biphenyl ] -4-yl) ethanone (2e)

2- (1- (3, 5-difluoro-3 ',4' -dimethoxy- [1,1' -biphenyl ] -4-yl) -1H-1,2, 3-triazole-4-yl) pyridine (2f)

3- (1- (2,2', 6-trifluoro-1, 1' -biphenyl ] -4-yl) -1H-1,2, 3-triazol-4-yl) pyridine (2g)

3- (1- (3'- (trifluoromethoxy) - [1,1' -biphenyl ] -4-yl) -1H-1,2, 3-triazole-4-yl) pyridine (2H)

2- (1- ([1,1' -biphenyl ] -4-yl) -1H-1,2, 3-triazol-4-yl) pyridine (2i)

Drawings

FIG. 1, chemical structures of azido synthetic blocks 1a to 1i used for the screening of active blocks in examples 1 and 2.

FIG. 2 shows fluorescence screening results of in-situ click chemistry coupled bacteria in-vitro transcription/translation system for azide synthesis of seven blocks 1 a-1 i by using escherichia coli (BL21) ribosome crude extract as a system matrix.

FIG. 3 shows fluorescence screening results of in-situ click chemistry coupled bacteria in-vitro transcription/translation system for azide synthesis of seven blocks 1 a-1 i by taking staphylococcus aureus (ATCC33591) ribosome crude extract as a system matrix.

FIG. 4 chemical structures of bacterial ribosome inhibitors 2c and 2h and other in situ click chemistry products (2a, 2b, 2 d-2 g, 2i) screened in example 1 and example 2.

Detailed Description

The screening method of the present invention is described in detail below with reference to examples: