CN111220811A - Screening method of TrpRS inhibitor - Google Patents

Abstract

The invention discloses a method for screening TrpRS inhibitor on protein level, which adopts an amino coupling method to fix a target protein TrpRS on the surface of a chip, prepares a compound to be screened into a proper concentration for single concentration or affinity screening, processes sensing signals uniformly to screen a compound with higher affinity with the TrpRS protein.

Description

Screening method of TrpRS inhibitor

Technical Field

The invention relates to the field of medicines, in particular to a screening method of an antibacterial medicine for inhibiting TrpRS protein activity.

Background

Bacterial resistance, especially multi-resistant gram-negative bacteria, has become a serious public health problem worldwide. Bacterial resistance threatens the life and health of human beings, and causes serious economic and social problems, and almost every year, people die due to antibiotic resistance. In 2015, the number of people dying due to bacterial drug resistance in the world is about 70 thousands, and according to the current growth rate, the number reaches 1000 thousands by 2050 years, which exceeds the number of people dying from cancer every year at present. Controlling antibiotic abuse, identifying new antibiotic targets, and developing new antibiotics have become the primary strategy for dealing with current drug-resistant infections.

Aminoacyl tRNA synthetases (aaRS) are the earliest proteins in the life evolution process, are widely present in archaea, eubacteria, eukaryotic cells, mitochondria, chloroplasts and other organelles, catalyze aminoacylation of amino acid and corresponding tRNA to realize accurate translation of genetic information, and play a crucial role in the biosynthesis of proteins. Once these enzymes are inhibited in the bacteria, biosynthesis of the protein is forced to terminate, inevitably resulting in a slow growth of the bacteria. aaRS are ubiquitous in cells, and although their catalytic functions are conserved, the aaRS of different species differ in structure, with major differences between prokaryotic and eukaryotic organisms. The different catalytic site structure of bacteria from human aaRS makes it possible to design specific aaRS inhibitors against microorganisms and helps to develop novel antibiotics targeting aaRS.

Currently 3 aaRS inhibitors have been approved as human or veterinary drugs, the IleRS inhibitor mupirocin (Bactroban), the pross inhibitor Halofuginone (HF) and the LeuRS inhibitor AN2690(Tavaborole), respectively. Clinical application of these drugs suggests that each enzyme in aaRS can be independently used as an important target for the development of novel antibiotics.

Tryptophan is an important amino acid for protein synthesis and plays an important role in the organism. TrpRS inhibitors include: innovative mycin, indomycin and the newly discovered TrpRS inhibitor with a novel structure which is screened by technical means such as high-throughput screening in recent years. The compounds act on the amino acid catalytic site of TrpRS, show better antibacterial activity, and are found in the traditional antibiotic discovery way based on activity screening, such as: the discovery of innovative mycin and indomycin has the defects of unclear target spots and the like in the early stage of research, so that the modification of the compound cannot be rationally carried out. In recent years, virtual screening methods have been developed mainly for single-site studies, and compounds obtained by screening are limited by selection and confirmation of pharmacophores and binding sites. The new structural compounds obtained by High Throughput Screening (HTS) are becoming important means for discovering new active compounds, but most of the compounds obtained by screening by the method are already 'formed' compounds, the relative molecular mass is large, the space for structural optimization is small, even part of 'excellent' binding groups are neglected due to the influence of a skeleton, and the difficulty in transforming the compounds into high-activity, high-selectivity and pharmaceutical molecules is large. Aiming at the problem of frequent drug resistance of the existing antibiotics, the selection of a novel target and the establishment of a rational screening model for screening a novel inhibitor are imminent.

Currently, there is no drug screening model targeting tryptophan tRNA synthetase (TrpRS).

Disclosure of Invention

In order to solve the above problems, the present invention provides a method for screening a TrpRS inhibitor.

The invention establishes a high-throughput screening model of the TrpRS inhibitor by utilizing iTC (isothermal titration calorimetry) technology and SPR (surface plasmon resonance) technology. The combination of the two technologies can quickly and effectively screen out candidate compounds for inhibiting the activity of the TrpRS protein.

The invention relates to a screening method of a TrpRS inhibitor, which uses TrpRS protein as a target and combines iTC (isothermal titration calorimetry) and SPR (surface plasmon resonance) for screening.

Further, the invention relates to a screening method of the TrpRS inhibitor, which comprises the following steps:

(1) establishing a stable isothermal titration calorimetry detection system, and determining the binding affinity (K) of TrpRS protein and positive compound_D) And the activity of the TrpRS protein;

(2) fixing TrpRS protein on SPR chip by amino coupling, passing positive compound solution and negative compound solution on the chip surface, detecting the binding signals of positive compound and TrpRS protein to obtain affinity K_DValue and affinity K to iTC in step (1)_DComparing the values, if K in step (1)_DThe value of K in step (2)_DIf the ratio of the values is between 1/20 and 2, confirming that the SPR screening model is established;

(3) respectively flowing a solution of a single-concentration positive compound and a solution of a compound to be screened through the surface of the chip, measuring a binding signal, comparing the binding signal of the compound to be screened and the TrpRS protein with the binding signal of the single-concentration positive compound and the TrpRS protein, and screening out a compound generating a binding signal which is more than 1/3 of the binding signal generated by the single-concentration positive compound;

(4) preparing the compound obtained by screening in the step (3) into a series of concentration solutions, and flowing the solutions through the surface of the chip to perform affinity K_DValue determination and affinity K with the positive compound determined in step (2)_DAnd comparing the values, and screening out the active compound with better binding force with the TrpRS protein.

According to the present invention, preferably, the positive compound is selected from one or more of the group consisting of innomycin, indomycin and tryptophan; the negative compound is selected from one or more of the group consisting of alanine, arginine, leucine, serine, glutamic acid, methionine and histidine.

According to the invention, in step (1), preferably, the affinity K of the TrpRS protein for binding to positive compounds is determined in the free state_DWhen the value is positive, the number of titration is 16; the titration volume of the 1 st drop is 0.4 muL, and the titration volumes of the rest drops are 2 muL; the equilibration time was 150 s. The concentration of the TrpRS protein solution and the concentration of the positive compound solution can be determined by one skilled in the art. Preferably, the buffer used for preparing the TrpRS protein solution and the positive compound solution is PBS or HBS buffer, and more preferably PBS buffer containing 5% DMSO.

According to the present invention, in the step (2), preferably, the positive compound and the negative compound are separately prepared as solutions of a series of concentrations to be flowed over the surface of the chip. One skilled in the art can set a range of concentrations, for example at least 5, preferably at least 6, most preferably 8 non-zero concentrations; the multiple between adjacent concentrations is, for example, 1.1 to 10 times, preferably 1.5 to 5 times, and most preferably 2 times. Preferably, the screening buffer used to prepare the positive compound solution and the negative compound solution is PBS or HBS buffer, more preferably PBS buffer containing 5% DMSO.

According to the invention, the positive compound obtained in step (2) is combined with the K of TrpRS_DThe value of K of TrpRS and the positive compound obtained in step (1)_DComparing the values if step (1) K_DAnd step (2) K_DThe SPR model is considered to be established if the ratio of (1/20) to (2), preferably (1/15) to (1), most preferably (1/10) to (1); if the ratio of the two is<1/20 or>2, adjusting the SPR experimental parameters.

According to the invention, in step (3), preferably, the positive compounds with the series of concentrations are run before screening, and the positive compounds with the series of concentrations are run again after screening is completed, so that the normal protein and instrument before and after screening are ensured. More preferably, single-concentration positive compounds and negative samples are used as quality control points for sample injection in the screening process; preferably, a single concentration of positive compound and negative sample is injected 10-30 times per assay of the compound to be screened, most preferably a single concentration of positive compound and negative sample is injected 20 times per assay of the compound to be screened.

According to the present invention, in step (3), preferably, the concentration of the single-concentration positive compound is 0.1 to 500. mu.M, more preferably 50 to 200. mu.M. Most preferably, the positive compound is innovated mycin at a concentration of 100. mu.M. The negative sample was screening buffer. Preferably, the screening buffer used for preparing the positive compound solution and the compound solution to be screened is PBS or HBS buffer, more preferably PBS buffer containing 5% DMSO.

According to the present invention, in step (4), the skilled person is able to set a range of concentrations, for example at least 5, preferably at least 6, most preferably 8 non-zero concentrations; the multiple between adjacent concentrations is, for example, 1.1 to 10 times, preferably 1.5 to 5 times, and most preferably 2 times. Preferably, the screening buffer used to prepare the compound solution to be screened is PBS or HBS buffer, more preferably PBS buffer containing 5% DMSO.

The invention takes TrpRS protein as a target, and establishes a high-throughput screening model for screening TrpRS inhibitors.

It is understood by those skilled in the art that although there is a difference in amino acid sequence of TrpRS proteins of different species, they can be immobilized on the surface of a chip as long as they contain a free primary amino group according to the principle of protein immobilization, and the difference in amino acid sequence does not affect the immobilization of proteins. The method of the present invention can be used to screen TrpRS protein inhibitors derived from various bacteria.

According to the invention, the TrpRS protein is preferably a Bacillus thermophilus TrpRS protein, and more preferably the amino acid sequence of the Bacillus thermophilus TrpRS protein is shown in SEQ ID NO.1, and the coding nucleotide sequence is shown in SEQ ID NO. 2.

According to the present invention, preferably, the SPR chip is a dextran chip (CM 5). Preferably, the chip is subjected to carboxyl activation treatment before coupling to the TrpRS protein. Preferably, EDC and NHS are used to activate carboxyl groups on the chip surface, more preferably EDC and NHS are used in a ratio of 1: 1. Preferably, the coupling buffer is a sodium acetate buffer, more preferably, the pH of the sodium acetate buffer is 5.5. Preferably, the concentration of TrpRS protein used for conjugation is at least 10. mu.g/ml, more preferably 20-30. mu.g/ml, most preferably 20. mu.g/ml. Preferably, the amount of TrpRS protein coupled is such that the response signal is at least 4000RU, more preferably between 4000 and 6000 RU. Preferably, the chip blocks excess carboxyl groups after coupling to the TrpRS protein. Preferably, the carboxyl group blocking is performed using ethanolamine. The method improves the coating technology of the SPR sensing chip and the components of the screening buffer solution, and obviously improves the screening signal.

The invention has the beneficial effects that:

1. the method can quickly, economically and high-flux determine the affinity between the drug to be screened and the target protein;

2. the invention can completely realize the mutual action determination of 'label-free' without marking the target protein;

3. the screening model is established on the protein level, the action sites of the compounds can be confirmed through competition experiments, the compounds are classified according to the difference of the action sites, and a basis is provided for the research of the later-stage structure-activity relationship;

4. the invention can evaluate the activity of TrpRS protein in real time to determine whether the activity is reduced;

5. the invention can eliminate some nonspecific adsorption to a great extent, and can avoid the false positive phenomenon caused by different molecular weights of the compounds through the modes of molecular weight correction and the like;

6. the screening method of the invention has higher sensitivity, can realize the determination of weak affinity, and can simultaneously meet the requirements of HTS (high throughput screening) and FBDD (fragment-based drug design).

7. The method improves the protein coating technology of the SPR sensing chip and the components of the screening buffer solution, saves the using amount of protein, only needs to fix the protein once in the whole screening process for a small and medium compound library (containing 1000-2000 compounds), can better keep the activity of the protein, and obviously improves the screening signal.

Drawings

iTC in example 1 of FIG. 1 measures raw data for a heat signal (FIG. 1A) and a heat integrated signal plot (FIG. 1B). After injection, the positive compound solution was titrated 16 times into the TrpRS solution in iTC cells, the area under each titration peak being equal to the total heat released by the titration. Plotting this integrated heat against the molar ratio of positive compound added to the pool, a complete binding isobologram of compound interaction with TrpRS can be obtained.

FIG. 2 the SPR signal changes during the pre-enrichment of the TrpRS protein coupled to the chip surface in example 2 were plotted continuously as time vs RU (resonance units) (FIG. 2-1), and the pre-enrichment experiments were performed using sodium acetate at pH5.5 and 5.0 as buffers, respectively, and the pre-enrichment results were not significantly different, thereby confirming that the buffer with a pH close to the isoelectric point of the protein, i.e., sodium acetate at pH5.5, was used as the buffer in the screening experiments. Results of pre-enrichment of TrpRS protein at various concentrations in example 2 (FIG. 2-2).

FIG. 3 changes in SPR signal during TrpRS protein conjugation in example 2 were plotted continuously as time vs RU (resonance units) and the course of the reaction was recorded. FIG. 3-1 is Fc3 channel (blank): EDC/NHS mixed solution activation-ethanolamine blocking. Fig. 3-2 is Fc4 channel (TrpRS protein coupled): EDC/NHS mixed solution activation-TrpRS protein coupling-ethanolamine blocking. The NTA method in example 2 fixed changes in SPR signal during protein coupling (FIGS. 3-3).

FIG. 4 changes in SPR signal resulting from binding of TrpRS by a series of concentrations of positive compound in example 2 were plotted as time against RU (resonance units) and the signal from the positive compound-protein interaction sensor was recorded at different concentrations (FIG. 4-1). Protein and positive compound interaction affinity curves fitted by software (fig. 4-2). FIGS. 4-3 and 4-4 are affinity curves obtained using HBS and Tris as buffers, respectively.

FIG. 5 comparison of the effect of the chip with TrpRS target protein in example 3 after the first use (Panel 1) and two months (Panel 2) (approximately 500 pins injection times) as a curve fitted by the software to the affinity of the interaction of the protein and the positive compound.

FIG. 6 sample screening scattergram in example 4.

Figure 7 verification of accuracy of screening models in example 4.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes or modifications can be made by those skilled in the art after reading the description of the present invention, and such equivalents also fall within the scope of the invention.

The principle of action of the screening method (screening model) of the present invention is as follows:

firstly, establishing a stable isothermal titration calorimetry detection system, and determining the binding affinity of the TrpRS protein and a positive compound and the activity of the TrpRS protein; fixing the TrpRS protein with determined activity on the surface of a chip, establishing an SPR model, and verifying the establishment of a screening model by using a positive compound; and (3) enabling the compound to be screened to flow through the chip, determining a positive screening index according to a screening result, and analyzing and processing the screening result.

Example 1 construction of a Stable isothermal titration calorimetry-iTC test System

Water dropping experiment: and (3) ensuring the cleanness of the sample pool and the sample injection needle, washing for 3 times by using deionized water, and performing a water-water titration experiment by using the deionized water as a sample. The instrument cleanliness status is determined from the baseline level thermal signal peak size, generally, signal peak heights within 0.02 signal indicate that the sample well and needle are clean.

Setting iTC parameters: preparing purified TrpRS protein (amino acid sequence is shown as SEQ ID NO.1, and coding nucleotide sequence is shown as SEQ ID NO. 2) of the thermophilic bacillus into solution by PBS buffer solution containing 5% DMSO; positive compounds (innovated mycin) were formulated in solution in PBS buffer containing 5% DMSO. Sucking positive compound (innovative mycin) solution into a sample injection needle, titrating TrpRS protein solution in a sample pool for multiple times with small volume, determining key parameters for obtaining an optimal titration curve,including number of titration drops, titration volume and equilibration time. This experiment determined that the number of titration drops was 16, the first drop had a titration volume of 0.4 μ L (the first drop was titrated with a small volume and was subtracted from the assay result because the insertion of the needle into the cell caused mixing of the tip ligand with the protein, which affected the first drop), and the other drops had a titration volume of 2 μ L. The equilibration time is the set interval after each titration is completed, which is intended to rebalance the system and ensure that the heat signal returns to baseline before the next titration. The experiment determined the interval to be 150 s. Other parameters: temperature: 25 ℃; the stirring time was 800 RPM; reference power is 9 μ cal.s^-1。

Preparing a sample, namely preparing the purified TrpRS protein (the amino acid sequence is shown as SEQ ID NO.1, and the coding nucleotide sequence is shown as SEQ ID NO. 2) of the thermophilic bacillus into a buffer system, wherein the buffer system is PBS buffer solution containing 5% DMSO, the total volume is 300 mu L, and the final concentration of the TrpRS is 49.34 mu M; preparing a positive compound (Innovamycin) into a solution by using a PBS (phosphate buffer solution) containing 5% DMSO, wherein the final concentration is 434.7 mu M, and the total volume is 500 mu L; the blank solution was PBS buffer containing 5% DMSO.

Blank control experiment: the injection needle was rinsed with buffer, 280. mu.L of blank solution was added to the sample cell with a Hamilton needle, 40. mu.L of the prepared 434.7. mu.M solution of the positive compound was automatically added to the injection needle, and aspiration was repeated several times to ensure that no air bubbles were present in the injection needle. Titration is carried out according to the set parameters.

Sample titration experiment: after blank control is titrated, three steps of deionized water cleaning, methanol rinsing and drying are carried out on the sample injection needle, and two steps of deionized water cleaning and blank solution rinsing are carried out on the sample pool in a manual mode. mu.L of 49.34. mu.M TrpRS protein solution was added to the cuvette using a Hamilton needle (careful addition to avoid air bubble formation), and 40. mu.L of the prepared 434.7. mu.M solution of the positive compound was automatically added to the needle and aspiration repeated several times to ensure that no air bubbles were present in the needle. Titration is carried out according to the set parameters.

And (3) data analysis: firstly, data is imported into Origin processing softwareAnd checking whether the original graph has abnormal signals caused by bubbles or other factors. Removing the first drop signal and abnormal signal and deducting blank, selecting 'oneset of sites' model fitting data, automatically presenting the affinity value result and information such as combination ratio by software, and obtaining fitting parameters after fitting, wherein the result is shown in figure 1. Affinity K of the positive compound to the TrpRS protein_D＝3.21×10^-6±324×10^-9M。

Example 2 establishment of SPR screening model

(1) Pre-enrichment of TrpRS protein

After flushing the system with 5% DMSO in PBS buffer, freshly prepared and filtered through a 0.22 μ M filter, the whole new chip was placed in the instrument.

The ability of TrpRS proteins to couple to the chip surface was tested prior to activation of the chip. TrpRS protein was prepared in 20. mu.g.ml using pH5.5, 5.0 sodium acetate buffers, respectively^-1The concentration of the protein and the pre-enrichment result of the protein are not obviously different, so that the pH buffer solution with the isoelectric point similar to that of the protein is adopted in the screening experiment, namely, the sodium acetate buffer solution with the pH of 5.5 is selected as the coupling buffer solution to be operated in the Fc4 channel of the chip. The results of the pre-enrichment are shown in FIG. 2-1.

The results of pre-enrichment of TrpRS protein at different concentrations were also compared in the experiment and are shown in FIG. 2-2. As can be seen, at 20. mu.g/ml, the pre-enrichment results were able to reach the expected response signal values (greater than 4000 RU). Therefore, the coupling is carried out by adopting the concentration, so that the experimental requirements can be met, and the consumption of the protein can be saved.

(2) Immobilization of TrpRS on chip

1) Immobilization of proteins by amino coupling on carboxyl-activated dextran chips (CM5)

Amino coupling reagent: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, EDC; n-hydroxysuccinimide, NHS.

With freshly prepared 1:1 EDC/NHS mixed solution activates carboxyl on the surface of the chip, and the flow rate is 10 mu L^-1At time 600s, the chip Fc3 and Fc4 channels were operated simultaneously, with Fc3 as the reference channel.

TrpRS protein was prepared in 20. mu.g.ml using sodium acetate buffer pH5.5^-1At a rate of 10. mu.L.min^-1The flow rate of the probe flows on the surface of the chip, the sample injection time is optimized, and the proper sample injection time is selected to ensure that TrpRS is coupled to the surface of the chip as much as possible (the response signal is more than 4000RU), and the probe operates on an Fc4 channel of the chip.

Blocking the activated excess carboxyl groups with ethanolamine at a flow rate of 10. mu.L.min^-1Time 600s, operated simultaneously on Fc3 and Fc4 channels, Fc3 as the reference channel. The results are shown in FIGS. 3-1 and 3-2.

2) Immobilization of proteins using NTA (nitrilotriacetic acid) chips

Comprises the steps of 1, washing the surface of a chip by 350mM EDTA to ensure that the chip is in a better state; 2. washing the surface of the chip by using a buffer solution to remove EDTA (ethylene diamine tetraacetic acid) remained on the surface, thereby preventing the subsequent nickel ion capture from being influenced; 3. sampling nickel ions; 4. injecting 3mM EDTA to wash the surface of the chip, and washing away the nickel ions which are not firmly combined; 5. TrpRS protein was formulated to 20. mu.g.ml^-1The solution of (a) is flowed over the chip surface to couple it to the chip surface. The results are shown in FIGS. 3-3. As can be seen, the response signal using NTA chip-immobilized protein was about 2000RU, while the response signal using CM5 chip-immobilized protein was 4000RU or more. Therefore, the CM5 chip is preferably used for protein immobilization in the present invention.

(3) Model building

Positive (Innovamycin) and negative (alanine) compounds were applied to the chip surface in 5% DMSO in PBS buffer at serial concentrations (0.7. mu.M, 1.4. mu.M, 2.8. mu.M, 5.6. mu.M, 11.3. mu.M, 22.6. mu.M, 45.25. mu.M, 90.5. mu.M) for 40s, 60s, 30. mu.l/min. The affinity results were analyzed by data processing software and the sensorgram curve obtained is shown in FIG. 4-1 and the fitted affinity curve is shown in FIG. 4-2. Affinity K of positive compound and TrpRS_DValue of 2.527X 10^-5M, results and affinity K determined by iTC experiment_DValue 3.21X 10^-6By comparison, it can be determined that the SPR model holds.

And (3) buffer comparison: positive ions were separated using HBS and Tris (pH7.4) as buffersThe sexual compound (innovated mycin) is prepared into the series of concentrations under the other conditions. The positive compound affinity curves are shown in FIGS. 4-3 and 4-4, respectively. Affinity K of positive compound and TrpRS_DThe values are 3.414X 10 respectively^-5M、4.98×10^-5M。

As can be seen, in the above three buffer systems, the affinity K is_DThe results of the value measurement are relatively similar and are all on the order of magnitude, so that all three buffer solutions are suitable for the affinity measurement and can be selected freely in theory, but because the protein immobilization method adopted by the invention is an amino coupling method, and the Tris buffer solution contains a large number of primary amino groups, the protein immobilization method can occupy activated carboxyl sites on the surface of a CM5 chip during protein coupling, so that the protein cannot be immobilized on the surface of the chip, and therefore when the Tris buffer solution is used as a screening buffer solution, the primary amino group-free buffer solution is required during protein coupling immobilization. Therefore, in the experiment, it is complicated to prepare a buffer solution containing no primary amino group such as PBS or HBS, immobilize the protein on the chip, and then prepare a Tris buffer solution to measure the affinity. The invention adopts PBS or HBS buffer solution to carry out protein coupling and affinity determination, thereby simplifying the experimental steps.

Example 3 model stability Studies

And (3) counting the prepared chip with the TrpRS target protein when the chip is used, and collecting data according to the use time and the sample injection times. Through statistics, the chip can keep 50% of the initial protein activity when the service time is two months and the sample injection frequency is about 500 needles, and the result comparison is shown in fig. 5. Although a reduction in the activity of the TrpRS protein results in a reduction in the signal generated by the interaction of the compound with the target, the affinity of the compound for the TrpRS target protein does not change in magnitude; and the positive compound and the negative compound are always selected, namely, signals generated by the interaction of all analyzed compounds including the positive compound and the target protein are reduced, and the data analysis of the experiment is based on comparing the signal generated by the TrpRS target combined with the selected compound and the positive compound respectively, so that the reduction of the overall signal intensity does not influence the experiment result, namely, the stability of the model is good.

Example 4: drug screening process

(1) And (3) sample clearing treatment:

taking an analyte library including a compound library, determining molecular weight information of an analyte, determining the concentration to be diluted, preparing the analyte into an analyte stock solution with a larger concentration by using DMSO as a solvent, and then diluting by using PBS buffer solution for 20 times to prepare an analysis solution.

The method comprises the steps of performing library cleaning treatment on an analyte to remove a sample with viscosity in a sample library, wherein protein does not need to be coupled on the surface of a chip during library cleaning treatment, and the method can be realized by using a blank channel of the chip. The sample injection time of each sample during the warehouse cleaning treatment is 30 s. In the experimental process, in order to avoid interference among samples, 50% DMSO is used for cleaning the pipelines after the injection of each needle is finished, and at the moment, the 50% DMSO solution does not flow through the surface of the chip, so that the viscous compound remained in the pipelines can be removed without influencing the surface of the chip.

(2) And (3) screening samples:

the positive compound was innovated mycin, which was prepared in a solution of 100. mu.M concentration in PBS buffer containing 5% DMSO. The compounds to be screened which had been subjected to the depot preparation were prepared into a 100. mu.M solution in 5% DMSO in PBS buffer. And respectively flowing a solution of a single-concentration positive compound and a solution of a compound to be screened through the surface of the chip, measuring a binding signal, and screening at a single concentration. And (3) operating the positive compound with the concentration gradient before screening, and operating the positive compound with the concentration gradient again after screening is finished, so that the protein and the instrument are normal before and after screening. In the screening process, a screening program carried by Biacore T200 is used for running a sequence, a single-concentration positive compound (innovating mycin: 100 mu M) and a negative sample (blank buffer solution) are used as quality control points for sample injection, and the sample injection is carried out once by 20 needles on average. The experimental flow scattergram is shown in fig. 6. In the two lines in the figure, the upper line is formed by connecting all sampling points of positive compounds in the screening process, the lower line is formed by connecting all sampling points of negative sample (blank buffer solution) substances in the screening process, and signals of the two lines are basically parallel to the horizontal axis, which shows that the instrument system and the protein have good stability in the screening process.

(3) And (4) screening results:

according to the signal size of each compound to be screened and the positive screening index (the signal generated by the 100 mu M innovative mycin of the single-concentration positive compound), compounds which generate signals higher than the positive screening index by 1/3 are selected, and are prepared into a series of solutions with concentration (0 mu M, 4.69 mu M, 9.375 mu M, 18.75 mu M, 37.5 mu M, 75 mu M, 150 mu M and 300 mu M) to flow through the surface of the chip to carry out affinity K_DAnd (4) measuring the value.

Performing statistics on the directionally synthesized compounds used in the experiment, and comparing the affinity of the obtained compounds with that of the positive compounds, when the affinity is greater than or equal to that of the positive compounds (i.e. K)_DThe value is less than or equal to 2.527 multiplied by 10^-5M) is defined as active compound. 18 active compounds with affinity greater than or equal to that of the positive compounds are obtained from the 73 compounds in the screening, and the screening positive rate is about 25%.

(4) Screening model accuracy validation

3 positive compounds were selected: indomycin, indomycin and tryptophan; 7 negative compounds: alanine, arginine, leucine, serine, glutamic acid, methionine, histidine. The results obtained by separately preparing the above 10 compounds into 100. mu.M solutions and sampling according to the screening process sequence (the positive compounds still throughout the screening process) are shown in FIG. 7 (wherein: 1: tryptophan; 2: Indomycin; 2: positive reference (Indomycin: 100. mu.M), 3: indolmycin; and the rest: negative compounds). It can be seen that the screening model is able to distinguish between positive and negative compounds very well.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> institute of medical and Biotechnology of Chinese academy of medical sciences

<120> method for screening TrpRS inhibitor

<160>2

<170>SIPOSequenceListing 1.0

<210>1

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<212>PRT

<213> TrpRS protein of Bacillus thermophilus ()

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Gly Asn Tyr Ile Gly Ala Leu Arg Gln Phe Val Glu Leu Gln His Glu

20 25 30

Tyr Asn Cys Tyr Phe Cys Ile Val Asp Gln His Ala Ile Thr Val Trp

35 40 45

Gln Asp Pro His Glu Leu Arg Gln Asn Ile Arg Arg Leu Ala Ala Leu

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Tyr Leu Ala Val Gly Ile Asp Pro Thr Gln Ala Thr Leu Phe Ile Gln

65 70 75 80

Ser Glu Val Pro Ala His Ala Gln Ala Ala Trp Met Leu Gln Cys Ile

85 90 95

Val Tyr Ile Gly Glu Leu Glu Arg Met Thr Gln Phe Lys Glu Lys Ser

100 105 110

Ala Gly Lys Glu Ala Val Ser Ala Gly Leu Leu Thr Tyr Pro Pro Leu

115 120 125

Met Ala Ala Asp Ile Leu Leu Tyr Asn Thr Asp Ile Val Pro Val Gly

130 135 140

Glu Asp Gln Lys Gln His Ile Glu Leu Thr Arg Asp Leu Ala Glu Arg

145 150 155 160

Phe Asn Lys Arg Tyr Gly Glu Leu Phe Thr Ile Pro Glu Ala Arg Ile

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Pro Lys Val Gly Ala Arg Ile Met Ser Leu Val Asp Pro Thr Lys Lys

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Met Ser Lys Ser Asp Pro Asn Pro Lys Ala Tyr Ile Thr Leu Leu Asp

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Asp Ala Lys Thr Ile Glu Lys Lys Ile Lys Ser Ala Val Thr Asp Ser

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Glu Gly Thr Ile Arg Tyr Asp Lys Glu Ala Lys Pro Gly Ile Ser Asn

225 230 235 240

Leu Leu Asn Ile Tyr Ser Thr Leu Ser Gly Gln Ser Ile Glu Glu Leu

245 250 255

Glu Arg Gln Tyr Glu Gly Lys Gly Tyr Gly Val Phe Lys Ala Asp Leu

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Ala Gln Val Val Ile Glu Thr Leu Arg Pro Ile Gln Glu Arg Tyr His

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His Trp Met Glu Ser Glu Glu Leu Asp Arg Val Leu Asp Glu Gly Ala

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Glu Lys Ala Asn Arg Val Ala Ser Glu Met Val Arg Lys Met Glu Gln

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Ala Met Gly Leu Gly Arg Arg Arg

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<212>DNA

<213> TrpRS gene of Bacillus caldovelox ()

<400>2

catatgggta tgaaaaccat ttttagcggc attcagccga gtggcgtgat caccatcggc 60

aattacattg gcgccctgcg ccagtttgtt gaactgcagc acgaatataa ctgctacttc 120

tgcatcgtgg atcagcacgc aattaccgtg tggcaagatc ctcatgaact gcgccagaat 180

attcgccgcc tggcagcctt atacctggcc gtgggtatcg acccgaccca ggcaaccctg 240

tttatccaga gcgaagttcc tgcccatgcc caggcagcct ggatgctgca gtgcatcgtg 300

tatatcggtg agttagagcg catgacccag ttcaaggaga agagcgcagg caaagaagcc 360

gtgagtgccg gtctgctgac atatccgccg ctgatggccg ccgatatcct gctgtacaat 420

accgatatcg tgccggtggg cgaggatcag aaacagcata tcgagctgac ccgcgatctg 480

gccgagcgct ttaataaacg ctacggcgag ctgtttacca ttccggaagc ccgcattccg 540

aaagtgggcg cccgtattat gagcctggtg gaccctacca agaagatgag caaaagcgac 600

ccgaatccga aggcctatat caccctgctg gacgacgcca aaaccattga gaagaaaatt 660

aaaagcgcag ttaccgacag cgagggtacc atccgctacg ataaagaggc caaaccgggc 720

atcagcaacc tgctgaacat ctacagcacc ctgagcggcc aaagtattga ggaactggag 780

cgccagtacg aaggcaaggg ctatggcgtg ttcaaagccg atctggccca ggtggtgatc 840

gaaaccctgc gcccgatcca ggagcgctac catcattgga tggaaagcga ggaactggac 900

cgtgtgctgg acgaaggtgc cgaaaaagcc aatcgtgtgg ccagcgaaat ggttcgcaaa 960

atggagcagg ctatgggtct gggtcgtcgc cgcctcgag 999

Claims (9)

1. A TrpRS inhibitor screening method is characterized in that the TrpRS protein is used as a target, and iTC (isothermal titration calorimetry) and SPR (surface plasmon resonance) are used in combination for screening.

2. The method for screening for a TrpRS inhibitor according to claim 1, which comprises the steps of:

(1) establishing a stable isothermal titration calorimetry detection system, and determining the binding affinity K of TrpRS protein and positive compound_DValues and activity of TrpRS protein;

(2) fixing TrpRS protein on SPR chip by amino coupling, passing positive compound solution and negative compound solution on the chip surface, detecting the binding signal of positive compound and TrpRS protein to obtain affinity K_DValue and affinity K to iTC in step (1)_DComparing the values, if K in step (1)_DThe value of K in step (2)_DThe ratio of the values is between 1/20 and 2, preferably between 1/15 and 1, and most preferably between 1/10 and 1, confirming that the SPR screening model is established;

(3) respectively flowing a solution of a single-concentration positive compound and a solution of a compound to be screened through the surface of the chip, measuring a binding signal, comparing the binding signal of the compound to be screened and the TrpRS protein with the binding signal of the single-concentration positive compound and the TrpRS protein, and screening out a compound generating a binding signal larger than 1/3 of the binding signal generated by the single-concentration positive compound;

(4) preparing the compound obtained by screening in the step (3) into a series of concentration solutions, and flowing the solutions through the surface of the chip to perform affinity K_DValue determination and affinity K with the positive compound determined in step (2)_DComparing the values, and screening out the activity with better binding force with TrpRS proteinA compound is provided.

3. The method for screening the TrpRS inhibitor according to claim 2, wherein the TrpRS protein has an amino acid sequence shown in SEQ ID NO. 1.

4. The screening method for TrpRS inhibitor according to claim 2 or 3, wherein the positive compound is selected from one or more of the group consisting of innomycin, indomycin and tryptophan; the negative compound is one or more selected from the group consisting of alanine, arginine, leucine, serine, glutamic acid, methionine and histidine.

5. The method for screening for a TrpRS inhibitor according to any one of claims 2 to 4, wherein the SPR chip is a CM5 chip.

6. The TrpRS inhibitor screening method of any one of claims 2-5 wherein the concentration of TrpRS protein used for conjugation in step (2) is at least 10 μ g/ml, preferably 20-30 μ g/ml, and most preferably 20 μ g/ml.

Preferably, the amount of TrpRS protein coupled is such that the response signal is at least 4000RU, most preferably between 4000 and 6000 RU.

Preferably, TrpRS protein is immobilized on CM5 chips using sodium acetate buffer at pH5.5 as coupling buffer.

7. The method for screening a TrpRS inhibitor according to any one of claims 2-6, wherein in step (2), the positive compound and the negative compound are prepared as solutions of a series of concentrations, respectively, and the solutions are passed over the chip surface.

Preferably, a solution of positive compounds and a solution of negative compounds are prepared using PBS buffer containing 5% DMSO.

8. The method for screening a TrpRS inhibitor according to any one of claims 2-7, wherein in step (3), the positive compounds at the series of concentrations are run before the screening, and the positive compounds at the series of concentrations are run again after the screening is completed.

Preferably, positive compounds and negative samples with single concentration are used as quality control points for sample injection in the screening process; more preferably, a single concentration of positive compound and negative sample is injected 10-30 times per assay of the compound to be screened, and most preferably a single concentration of positive compound and negative sample is injected 20 times per assay of the compound to be screened.

9. The TrpRS inhibitor screening method of any one of claims 2-8 wherein 100. mu.M Innovamycin is used as a single positive concentration compound in step (3).

Preferably, the negative sample is a screening buffer.

Preferably, the screening buffer used for preparing the positive compound solution and the compound solution to be screened is a PBS buffer containing 5% DMSO.

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