Small molecule inhibitors targeting DksA and their applications

Abstract

The present invention provides a small molecule inhibitor for targeted inhibition of DksA and its application. The small molecule inhibitor for targeted inhibition of DksA can be used in the preparation of drugs for treating Escherichia coli disease and salmonellosis. The present invention uses high-throughput virtual screening technology to screen out 9 small molecule inhibitors for targeted inhibition of DksA, which have the advantages of wide application range and strong targeting. It has antibacterial effect on a variety of Escherichia coli and Salmonella, and affects bacterial biofilm formation and antibiotic resistance. Small molecule inhibitors for targeted inhibition of DksA have played an important role in the field of infection treatment of foodborne diseases caused by Escherichia coli and Salmonella, and provide new targeted therapeutic drugs for the control of clinical Escherichia coli disease and salmonellosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and molecular technology, and specifically relates to a small molecule inhibitor for targeted inhibition of DksA and its application. Background Art

[0002] Foodborne pathogens have always been closely related to food safety and public health issues that have attracted widespread international attention. According to statistics, in bacterial food poisoning incidents in various countries around the world, the problem of bacterial resistance is becoming increasingly serious. The number of deaths caused by infection with resistant bacteria is about 700,000 per year worldwide, and it is estimated that this number may reach 10 million by 2050. The serious problem of drug resistance has brought severe challenges to humans in the fields of medical clinics, veterinary clinics, food animal breeding and food safety. At present, drug resistance of animal-derived bacteria is very common in my country, and multi-drug resistant and even pan-drug resistant strains continue to appear, seriously endangering food safety and public health.

[0003] Salmonellosis is an infectious disease caused by Salmonella infection that affects humans and a variety of animals, causing serious harm to the breeding industry. In addition, the increasing infection rate in the human population has also made Salmonellosis a public health problem that affects the world. Due to the diversity of O and H antigens, Salmonella has a rich serotype, which currently exceeds 2,600 species. Salmonella Typhimurium and Salmonella enteritidis in the enteric subspecies of Salmonella enterica are the two major serotypes that cause salmonellosis in humans and animals. Salmonella Typhimurium is the most common serotype that causes foodborne diseases, which can cause acute gastroenteritis in humans and can cause systemic typhoid fever in mice. As a facultative intracellular parasite, Salmonella proliferates and spreads in cells by forming endosomes that encapsulate the bacteria. Salmonella can cause disease in a variety of animals, causing latent infection and persistent infection in adult animals that can last for several months, which brings great difficulties to the prevention and treatment of the disease. Globally, Salmonella causes about 115 million illnesses and about 370,000 deaths each year. Among bacterial food poisoning cases in my country, 70-80% are caused by Salmonella infection. In addition, the types of drug-resistant genes carried by Salmonella are increasing, and drug resistance is increasing. The cross-transfer of drug-resistant genes between farmed animals, the environment, and humans has caused serious harm to public health and food safety.

[0004] Escherichia coli, as a symbiotic bacterium in the human intestine, is mostly non-pathogenic, but when it captures pathogenic or virulence factors from the outside world, it will transform into pathogenic Escherichia coli, which can cause a variety of zoonotic diseases. Escherichia coli infection in farmed animals will not only lead to increased animal morbidity and mortality, but also reduce egg production in poultry, deteriorate the meat quality of livestock and poultry, and increase breeding and treatment costs. Infection with pathogenic Escherichia coli can cause diseases such as calf diarrhea, hemorrhagic enteritis and sepsis. Avian pathogenic Escherichia coli can also cause local or systemic infection in poultry. Such infections not only cause huge economic losses to animal breeding, but once they infect humans or pollute the surrounding environment through excrement, they will indirectly infect humans and other animals, causing serious harm to food safety, public health and human health.

[0005] Since the 1940s, antimicrobial drugs such as natural and chemically synthesized antibiotics have been considered a panacea for treating infectious diseases, greatly improving people's quality of life and significantly reducing the mortality rate caused by bacterial infections. However, with the extensive use and abuse of antimicrobial drugs in clinical practice, multidrug-resistant strains continue to emerge and become widely prevalent and spread around the world, posing a serious threat to human health. In particular, there are fewer and fewer effective therapeutic drugs available for infections caused by Gram-negative bacteria such as Enterobacter, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii.

[0006] In Escherichia coli and Salmonella, the RNA polymerase-binding transcription factor DksA is a pleiotropic regulator that controls bacterial metabolism and virulence under non-strict growth conditions. However, few small molecule inhibitors targeting DksA have been reported. Summary of the invention

[0007] The present invention aims to provide a small molecule inhibitor and application thereof for targeting inhibition of DksA in view of the deficiencies in the prior art.

[0008] To achieve the above object, the solution of the present invention is:

[0009] In one aspect, a small molecule inhibitor targeting DksA is provided, wherein the structure of the small molecule inhibitor is any one of the following structures:

[0010]

[0011] On the other hand, provided is a use of the above-mentioned small molecule inhibitor targeting and inhibiting DksA in the preparation of a drug for treating Escherichia coli disease and Salmonellosis.

[0012] Preferably, the amino acid sequence of DksA is (SEQ ID NO.1):

[0013] MQEGQNRKTSSLSILAIAGVEPYQEKPGEEYMNEAQLAHFRRILEAWRNQLRDEVDRTVTHMQDEAANFPDPVDRAAQEEEFSLELRNRDRERKLIKKIEKTLKKVEDEDFGYCESCGVEIGIRRLEARPTADLCIDCKTLAEIREKQMAG.

[0014] On the other hand, a drug for treating Escherichia coli disease and Salmonellosis is provided, wherein the active ingredient of the drug is a small molecule inhibitor that targets and inhibits DksA.

[0015] Preferably, the drug is used to target and inhibit DksA.

[0016] Preferably, the medicament further comprises a pharmaceutically acceptable carrier.

[0017] Due to the adoption of the above scheme, the beneficial effects of the present invention are:

[0018] The present invention uses high-throughput virtual screening technology to screen out 9 small molecule inhibitors that target and inhibit DksA, which have the advantages of wide application range and strong targeting. They have antibacterial effects on a variety of Escherichia coli and Salmonella, and have an impact on bacterial biofilm formation and antibiotic resistance. Small molecule inhibitors targeting DksA have played an important role in the field of infection treatment of foodborne diseases caused by Escherichia coli and Salmonella, and provide new targeted therapeutic drugs for the control of clinical Escherichia coli and Salmonellosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a 3D structural analysis diagram of DksA in Example 1 of the present invention (wherein, Figure A is the DksA structure obtained based on the 7KHE model, Figure B is the interaction between ppGpp (globular) and three amino acids in DksA (based on the 7KHE model), and Figure C / D is the molecular superposition diagram and RMSD value of the AF model, 1TJL and 7KHE model).

[0020] Figure 2 It is the molecular docking pocket and analysis diagram of the DksA protein in Example 1 of the present invention (wherein, pocket: refers to the small molecule binding pocket on the DksA protein. Through molecular docking, it is shown that there are two docking pockets on the protein, namely pocket 1 and pocket 2; Size: the size of the small molecule binding pocket; PLB: PLB means phosphate receptor, which refers to the protein ligand binding affinity, arranged in descending order, and the pocket position is generally determined by the actual pocket position and the PLB value; Hyd: pocket quality score; Side: refers to the site of the pocket; Residues: contains amino acid site information).

[0021] Figure 3 It is a scoring function diagram of Drug Score and Lipinski Score in Example 1 of the present invention (wherein, logS: water solubility; HIA: oral availability; logP: lipid solubility; hERG pIC50: half inhibition concentration of cardiotoxicity; 2D6 affinitycategory: drug interaction risk index, referring to cytochrome P450 enzyme metabolism and P protein (P-gp category) absorption effect; 2C9 pKi: 2C9 protein kinase inhibitor; P-gp category: P protein; PPB90: antibacterial drug binding rate to plasma protein; BBB category: blood-brain barrier; HBA: number of hydrogen bond acceptors; HBD: number of hydrogen bond donors; MW: molecular weight).

[0022] Figure 4 This is an analysis chart of the inhibitory effects of 9 compounds and 2 control drugs (Norfloxacin and Enrofloxacin) in Example 3 of the present invention on 5 strains.

[0023] Figure 5 OD values ​​after crystal violet staining after the 9 compounds and 2 control drugs (Norfloxacin and Enrofloxacin) in Example 5 of the present invention acted on the 5 strains 620nm picture.

[0024] Figure 6 The crystal violet staining results of the nine compounds and two control drugs (Norfloxacin and Enrofloxacin) in Example 6 of the present invention after acting on five strains, respectively.

[0025] Figure 7 This is a diagram showing the interaction pattern between compound No. 5T13748 in Example 6 of the present invention and DksA protein.

[0026] Figure 8 This is a diagram showing the interaction pattern between compound No. 6T6822 in Example 6 of the present invention and DksA protein.

[0027] Fig. 9 This is a diagram showing the interaction pattern between compound No. 7T7449 in Example 6 of the present invention and DksA protein.

[0028] Fig.10 This is a diagram showing the interaction pattern between compound No. 8T7845 and DksA protein in Example 6 of the present invention.

[0029] Fig.11 This is a diagram showing the interaction pattern between compound No. 10T8854 and DksA protein in Example 6 of the present invention.

[0030] Fig.12 This is a diagram showing the interaction pattern between compound No. 12T7691 in Example 6 of the present invention and DksA protein.

[0031] Fig.13 This is a diagram showing the interaction pattern between compound No. 19T4434 and DksA protein in Example 6 of the present invention.

[0032] Fig.14 This is a diagram showing the interaction pattern between compound No. 25T2525 in Example 6 of the present invention and DksA protein.

[0033] Fig.15 This is a diagram showing the interaction pattern between compound No. 40T9285 and DksA protein in Example 6 of the present invention. DETAILED DESCRIPTION

[0034] The present invention provides a small molecule inhibitor for targeted inhibition of DksA and application thereof.

[0035] DksA is a conserved RNA polymerase binding protein that is commonly found in many gastrointestinal pathogens, such as Escherichia coli, Salmonella, and Proteus. Studies have shown that DksA plays an important regulatory role in the development of bacterial resistance and pathogenicity. It exerts a global regulatory effect by binding to RNA polymerase, blocking the interaction between polymerase and DNA, inhibiting the formation of the initiation complex, enhancing the ppGpp effect, and initiating the transcription of NTP on rRNA. DksA is involved in a variety of biological functions, such as negative regulation of rRNA expression; inhibition of transcript elongation; inhibition of exonucleoside RNA cleavage; inhibition of pyrophosphorylation; and increase of intrinsic termination. Studies have shown that DksA is involved in the regulation of bacterial motility and biofilm formation, and promotes the invasion of Salmonella typhimurium into epithelial cells by positively regulating the SPI-1 gene. In addition, DksA expressed during intestinal infection is necessary for pathogenic bacteria to colonize the gastrointestinal tract and cause systemic infection. Therefore, the present invention aims to screen novel small molecule inhibitors that specifically inhibit DksA, in order to obtain targeted therapeutic drugs for the treatment of Escherichia coli disease and Salmonellosis. It is expected that the antibacterial effect of the compound will be superior to the sensitivity or activity of other similar antibiotics, with extremely low toxic side effects on the animal itself and its environment, and will not easily develop drug resistance.

[0036] Embodiment 1:

[0037] 1. Analysis of DksA protein structure: It consists of 151 amino acids and contains a zinc finger structure, in which four cysteines are involved in the binding of metal zinc ions (C114, C117, C135, C138). The 3D structure of DksA protein is shown in Figure 1As shown in Figure A, the DksA protein consists of an irregular turn at the N-terminus, a G domain consisting of two long α-helices, CCtip and a C-terminal α-helix. Figure 1 Figure B shows that DksA binds to the second channel of RNA polymerase. The regions involved in the interaction are the G domain (helix), CC tip, and C-terminal α helix. When conducting structural analysis, two models from crystal structure and cryo-EM structure (1TJL and 7KHE) were selected at the same time, and their 3D structure (AF model) was constructed using AlphaFold. The superposition of these three models shows that their RMSD values ​​are all 0.00, and their structures are highly similar ( Figure 1 Figure C and D in the figure), which means the stability of the structure in different states. RMSD is the root mean square deviation (Root Mean Square Deviation, RMSD), and the definition of RMSD is as follows:

[0038]

[0039] Where N is the number of atoms, m i is the mass of atom i, X i is the coordinate vector of target atom i, Y i is the coordinate vector of the reference atom i, and M is the total mass. When RMSD is not mass-weighted, all m i = 1 and M = N. In Amber, the unit of RMSD is Angstrom. ).

[0040] In molecular simulation, RMSD is used to measure the degree of conformational difference or trajectory stability. In molecular docking, the ligand crystallized on the protein is often extracted, docked back using docking software, and then the RMSD values ​​between the two ligands before and after docking are compared. The smaller the value, the higher the accuracy of the docking software. RMSD = 0.0 means perfect overlap. The larger the RMSD, the greater the deviation of the target molecule from the reference molecule and the more unstable the structure.

[0041] 2. High-throughput virtual screening of small molecule inhibitors targeting DksA:

[0042] (1) Molecular docking: The compound library for this screening is the targetmol compound library (T001). The compound library contains 38,969 compounds. The compound library was subjected to molecular cleaning to remove salt ions, metal ions, small fragments, etc. The 3D structure of the compound was obtained through energy minimization. In order to ensure the global conformation of small molecule compounds during the virtual screening process, multiple conformations of small molecule compounds were generated. The virtual screening software FRED (Ver 3.2.0.2) was used to perform batch molecular docking of each conformation in the established target screening area to obtain the affinity score and interaction mode of each compound with three different DksA protein models.

[0043] The SiteFinder plug-in in MOE was used to detect the possible small molecule binding pockets on the surface of these models. It was found that the surfaces of these three models all contain two small molecule binding pockets. The positions of these pockets and their properties (size, number of hydrophobic atoms, pocket quality score, amino acid content, etc.) are as follows: Figure 2 As shown, pocket1 in all three models showed better properties than pocket2 (such as PLB value). Although the structures of the three models are very similar, their pocket sizes, properties and amino acid sites are also different. In order to maximize the hit rate of the screening compounds, the three models were screened based on the Pocket1 region at the same time during this screening process. The pockets Pocket1 and Pocket2 of the AF model are adjacent, and Pocket2 contains the ppGpp binding site (K98, R129, K139). Therefore, the Pocket1 and Pocket2 regions of the model can be combined into one for molecular screening.

[0044] (2) Compound screening: The compounds were screened for drugability properties, and 340 compounds were screened. The properties of small molecule compounds were calculated and analyzed using Stardrop software (Version 6.5.0), such as water solubility (logS), lipid-water distribution coefficient (logP), molecular weight, molecular flexibility, hydrogen bonding properties, surface accessible area (TPSA), CYP2C9 enzyme degradation level (CYP2C9 is an enzyme in human metabolism, a membrane-bound hemoglobin, which can hydroxylate and metabolize many drugs of different properties, mainly acidic substrates. About 16% of clinical drugs are metabolized by CYP2C9), hERG inhibition rate index (hERG: human ether-a-go-go related gene, potassium ion channel and human EAG related gene, hERG inhibition problem is still the most direct and effective strategy to improve cardiac toxicity), oral availability (HIA), drug interaction risk (2D6) and other indicators. The 2D6 index includes the effects of cytochrome P450 enzyme metabolism and P protein absorption. Evaluation, scoring and screening results are shown in the Drug Score using the screening criteria for oral non-CNS drugs. Figure 3 . The scoring function is a mathematical statistical result of the properties described above. The score ranges from 0 to 1. The higher the value, the better the drugability of the compound. 40 compounds with high scores and good drugability were screened out, and these compounds were preliminarily screened for inherent antibacterial properties against standard strains of Escherichia coli and Salmonella and multiple clinical isolates. The Lipinski rule is used for the initial screening of the compound database in order to eliminate those molecules that are not suitable for becoming drugs, narrow the scope of screening and reduce the cost of drug development. LipinskiScore refers to the five principles of drug-like drugs, called Lipinski rules, which are as follows: A small molecule drug must have the following properties: molecular weight less than 500; number of hydrogen bond donors less than 5; number of hydrogen bond acceptors less than 10; lipid-water partition coefficient less than 5; number of rotatable bonds not exceeding 10. Compounds that meet the Lipinski Score principle will have better pharmacokinetic properties and higher bioavailability during metabolism in the body.

[0045] Embodiment 2:

[0046] Analysis of antibiotic resistance of Escherichia coli and Salmonella

[0047] 27 commonly used antibiotics were selected for drug sensitivity test, and the drug resistance of Escherichia coli standard strain (E), Salmonella standard strain (ST) and clinical isolates of Escherichia coli (E-10), Salmonella (ST-477), Salmonella (ST-3), Salmonella (ST-7), Salmonella (ST-6), Salmonella (ST-1) and Salmonella (ST-71) were analyzed by paper strip method. Single colonies of each strain were picked and added to 1 mL of LB, shaken at 37°C, 180 rpm overnight, and expanded to OD 1:100 the next day. 620nm The concentration of the bacterial solution was adjusted to 0.5 McFarland turbidity with LB medium, 100 μL of each bacterial solution was dropped onto the LB plate, coated with a glass rod, and allowed to stand for 15 minutes before attaching a drug-sensitive sheet. The plate was cultured in a 37°C incubator for 16-18 hours, the diameter of the inhibition zone was measured (as shown in Table 1), and the number of antibiotics to which each strain was sensitive was counted.

[0048] Table 1 Results of sensitivity test of 9 strains to 27 antibiotics

[0049]

[0050]

[0051] Note: S stands for sensitive, I stands for intermediately sensitive, and R stands for resistant

[0052] Through analysis of the data in Table 1, standard strains of Escherichia coli (resistant to 11 antibiotics) and Salmonella (resistant to 12 antibiotics), as well as highly resistant clinical isolates of Escherichia coli E-10 (resistant to 18 antibiotics), Salmonella ST-7 (resistant to 14 antibiotics) and Salmonella ST-6 (resistant to 14 antibiotics) were selected for subsequent inherent antibacterial tests.

[0053] Embodiment 3:

[0054] Intrinsic antibacterial assay of small molecule inhibitor compounds

[0055] Preparation of compound stock solutions: 40 compounds were dissolved in dimethyl sulfoxide (DMSO) according to their different solubilities to form stock solutions. According to the preliminary experiment, the compound stock solutions were diluted with LB medium to a working solution with a concentration of 800 μM. Norfloxacin and enrofloxacin were selected as control compounds. Norfloxacin was dissolved in dimethyl sulfoxide (DMSO) to form a 10 mM stock solution, and the stock solution was diluted with LB medium to form a working solution with a concentration of 800 μM; enrofloxacin hydrochloride was dissolved in water to form a 10 mM stock solution, and the stock solution was diluted with LB medium to form a working solution with a concentration of 800 μM.

[0056] Escherichia coli (E) and Salmonella (ST), clinically isolated Escherichia coli (E-10), Salmonella (ST-6), and Salmonella (ST-7) were cultured to OD 620nm The concentration of bacteria was 0.5, and the bacterial solution was diluted with LB to 1x10 5 CFU / mL, 40 compounds and 2 control drugs (concentration of 800 μM) were cultured at 37°C for 24 h and then OD was measured 620nm The number of colonies was calculated by the dilution plating method, and the inhibition rate of each compound on bacteria was statistically analyzed.

[0057] The number of colonies in the control group = the average number of colonies without adding small molecule inhibitors

[0058] The number of colonies in the experimental group = the average number of colonies after adding small molecule inhibitors

[0059] Bacterial inhibition rate = (number of colonies in the control group - number of colonies in the experimental group) * 100% / number of colonies in the control group

[0060] Based on the above calculation formula, the inhibition rates of 40 small molecule inhibitors and 2 control drugs were calculated and shown in Tables 2, 3, 4, 5 and 6.

[0061] Among them, the structures of 9 small molecule inhibitors are any of the following structures:

[0062]

[0063] Table 2 Inhibition rates of 40 small molecule inhibitors and 2 control drugs against Escherichia coli (E)

[0064]

[0065] It can be seen from Table 2 that 9 of the 40 small molecule inhibitors have excellent antibacterial effects. The inhibition rates of Escherichia coli (E) exceeding 50% include NO.5 (inhibition rate 51.23%), NO.6 (inhibition rate 60.01%), NO.7 (inhibition rate 90.13%), NO.8 (inhibition rate 66.35%), NO.10 (inhibition rate 91.57%), NO.12 (inhibition rate 79.19%), NO.19 (inhibition rate 89.16%), NO.25 (inhibition rate 89.74%) and NO.40 (inhibition rate 54.12%), and the control drugs Norfloxacin (inhibition rate 84.66%) and Enrofloxacin (inhibition rate 80.46%).

[0066] Table 3 Inhibition rates of 40 small molecule inhibitors and 2 control drugs on Escherichia coli (E-10)

[0067]

[0068] It can be seen from Table 3 that the inhibition rates of Escherichia coli (E-10) exceeding 50% include NO.5 (inhibition rate 52.66%), NO.6 (inhibition rate 60.28%), NO.7 (inhibition rate 91.25%), NO.8 (inhibition rate 69.22%), NO.10 (inhibition rate 81.23%), NO.12 (inhibition rate 74.25%), NO.19 (inhibition rate 89.55%), NO.25 (inhibition rate 85.22%) and NO.40 (inhibition rate 51.26%), and the control drugs Norfloxacin (inhibition rate 74.88%) and Enrofloxacin (inhibition rate 90.01%).

[0069] Table 4 Inhibition rates of 40 small molecule inhibitors and 2 control drugs against Salmonella (ST)

[0070]

[0071] It can be seen from Table 4 that the inhibition rates of NO.5 (76.38%), NO.6 (65.24%), NO.7 (94.56%), NO.8 (74.85%), NO.10 (95.63%), NO.12 (77.78%), NO.19 (94.58%), NO.25 (98.24%) and NO.40 (50.01%) against Salmonella (ST) are over 50%, and the control drugs are Norfloxacin (82.47%) and Enrofloxacin (77.56%).

[0072] Table 5 Inhibition rates of 40 small molecule inhibitors and 2 control drugs against Salmonella (ST-6)

[0073]

[0074] It can be seen from Table 5 that the inhibition rates of NO.5 (54.27%), NO.6 (61.57%), NO.7 (90.48%), NO.8 (70.21%), NO.10 (67.22%), NO.12 (72.13%), NO.19 (91.27%), NO.25 (91.21%) and NO.40 (50.58%) against Salmonella ST-6 are over 50%, and the control drugs are Norfloxacin (88.10%) and Enrofloxacin (86.42%).

[0075] Table 6 Inhibition rates of 40 small molecule inhibitors and 2 control drugs against Salmonella (ST-7)

[0076]

[0077] It can be seen from Table 6 that the inhibition rates of Salmonella (ST-7) exceeding 50% include NO.5 (inhibition rate 51.02%), NO.6 (inhibition rate 60.14%), NO.7 (inhibition rate 94.33%), NO.8 (inhibition rate 65.11%), NO.10 (inhibition rate 89.66%), NO.12 (inhibition rate 79.11%), NO.19 (inhibition rate 90.06%), NO.25 (inhibition rate 90.24%) and NO.40 (inhibition rate 50.44%), and the control drugs Norfloxacin (inhibition rate 85.93%) and Enrofloxacin (inhibition rate 78.43%).

[0078] In summary, the standard of small molecule inhibitors with an inhibition rate of more than 50% at a final concentration of 400 μM was used to screen small molecule inhibitors with better activity in the targeted inhibition of DksA. The results of the 9 small molecule inhibitors with an inhibition rate of more than 50% on bacteria at a final concentration of 400 μM were plotted as follows: Figure 4 As shown, compound 5 has the highest inhibition rate against Salmonella (76.38%); compound 6 has a comparable inhibition rate against the five bacteria, which is around 61.44%; compound 7 has an inhibition rate of more than 90% against the five strains; compound 8 has a highest inhibition rate against ST, which is 74.85%; compound 10 has a highest inhibition rate against ST, which is 95.63%; compound 12 has comparable inhibition rates against Escherichia coli E (inhibition rate of 79.19%) and Salmonella ST-7 (inhibition rate of 79.11%); compound 19 has a highest inhibition rate against Salmonella ST, which is 94.58%; compound 25 has a highest inhibition rate against Salmonella ST, which is 98.24%; compound 40 has a relatively average inhibition rate against the five strains, which is around 51.28%; the control drug Norfloxacin has a highest inhibition rate against Salmonella ST-6, which is 88.16%, and the control drug Enrofloxacin has a highest inhibition rate against Escherichia coli E-10, which is 90.01%.

[0079] Embodiment 4:

[0080] Antimicrobial sensitization test with small molecule inhibitors

[0081] The standard strain of Salmonella ST was selected for antibacterial sensitization test. A single colony of Salmonella ST was added to 1 mL of LB and shaken at 37°C and 180 rpm overnight. The next day, the OD of the culture was expanded to 1:100. 620nm is 0.5, and the bacterial solution concentration is adjusted to 1x10 5CFU / mL, according to the test data in Table 4, the antibacterial rate of the compound against Salmonella ST is about 65.00%, 9 compounds with different final concentrations are added and cultured overnight at 37°C, 100 μL of each bacterial solution is dropped on the LB plate and coated with a glass rod, and then placed on the drug sensitive sheet after standing for 15 minutes, and cultured in a 37°C incubator for 16-18 hours to measure the diameter of the antibacterial zone. The results are shown in Table 7.

[0082] Table 7 Antibacterial sensitization results of 9 small molecule inhibitors on Salmonella ST

[0083]

[0084] As shown in Table 7, compound No. 6 enhances the antibacterial effect of gentamicin on Salmonella ST, and compound No. 12 enhances the antibacterial effect of tetracycline on Salmonella ST.

[0085] Embodiment 5:

[0086] Effects of small molecule inhibitors on biofilm

[0087] Biofilm significantly enhances the pathogenicity of Salmonella typhimurium to mice. The extracellular matrix of Salmonella biofilm is mainly composed of curly pili, cellulose, BapA (biofilm-associated protein), O capsule antigen, lipopolysaccharide (LPS), extracellular DNA (eDNA), etc. Crystal violet is a basic dye that can bind to DNA in the cell nucleus, thereby staining the cell nucleus. The crystal violet staining method can quantify the ability of the test bacteria to form biofilm.

[0088] The standard strains of Escherichia coli (E) and Salmonella (ST), clinically isolated Escherichia coli (E-10), Salmonella (ST-6), and Salmonella (ST-7) were cultured to OD 620nm The concentration of the bacterial solution was 1x10 5 CFU / mL, take 200μL of the diluted bacterial solution and inoculate it into a 96-well culture plate, repeat 3 replicates for each strain, use the culture medium as the blank control, use the bacterial culture as the positive control, and culture at 37℃ for 24h. After the culture is completed, discard the culture medium, rinse with PBS 3 times to wash away impurities and floating bacteria, then add 200μL of methanol to each well to fix the biofilm, discard the methanol after 15min, dry it, add 200μL of 0.1% crystal violet to stain for 5min, wash off the excess dye, dry it, add 200μL of 33% glacial acetic acid to each well, and measure the OD after high-speed oscillation of the microplate reader for 20min 620nm, the blank wells without inoculated strains were stained and used as blank controls. The OD value reflects the adhesion strength of the biofilm to the contact surface. According to the critical ODc value (the OD value obtained by adding three times the standard deviation of the blank wells to the average value of the blank wells), the biofilms can be classified as follows: OD ≤ ODc indicates non - adhesion (0), ODc < OD ≤ 2ODc indicates weak adhesion (+), 2ODc < OD ≤ 4ODc indicates medium adhesion (++), and OD > 4ODc indicates strong adhesion (+++). The OD values of the 9 compounds on 5 tested strains after crystal violet staining 620nm As Figure 5 shown, after the action of compounds 7, 12, 19, 25 and the control drugs (Norfloxacin and Enrofloxacin), the biofilms of the strains were weakly adherent. After the action of compounds 5, 6, 8, 10 and 40, the biofilms of the strains were moderately adherent. The positive control biofilms of each strain were moderately adherent, but the amount of biofilm was greater than that of the strains added with compounds. The crystal violet staining photos are as Figure 6 shown. The darker color indicates a larger amount of biofilm, indicating a larger amount of bacteria and a smaller antibacterial rate of the small - molecule inhibitor.

[0089] Calculation formula:

[0090] where is the average value of x1, x2 and x3 of the blank wells, and n is the number of samples.

[0091] Example 6:

[0092] Interaction modes of 9 compounds with DksA protein

[0093] After the above - mentioned experiments, the interaction mode diagrams of 9 compounds with the target were predicted. Through high - throughput virtual screening, based on the 3D structure model AF of DksA, after protonation treatment and structure optimization, the small - molecule compound binding pockets on its surface were analyzed. The corresponding small - molecule compound binding pockets in each model were determined as the docking regions for virtual screening. The compound library file with multiple conformations was screened using the OEDock software of OpenEye company, and the Chemgauss4 scoring function was used to evaluate the affinity of each compound with the target. Finally, the affinity of the compounds and the molecular docking models of their complexes with the protein target were obtained, and the druggability properties of these compounds were analyzed. Further screening of suitable compounds was carried out through affinity, druggability properties, etc., and 9 small - molecule inhibitors with better activity targeting and inhibiting DksA were screened out. These 9 compounds all interact with the amino acids in the molecular pocket of DksA and do not interact with the ppGpp binding site. The results are as Figure 7 , Figure 8 , Fig. 9 , Fig.10 , Fig.11 , Fig.12 , Fig.13 , Fig.14 and Fig.15 shown.

[0094] Select compounds NO.5, NO.6, NO.7, NO.8, NO.10, NO.12, NO.19, NO.25 and NO.40, and observe their affinity scores with DksA. The affinity values ​​in the AF model and the fact that these compounds are all bound to the pocket2 region, with which amino acids the compounds form hydrogen bond interactions and π hydrogen bond interactions, are mainly surrounded by positively charged amino acids. These 9 compounds have relatively ideal affinity scores and good drugability properties. Figure 7 The interaction pattern of compound No. 5T13748 and DksA protein: A, the complex structure of DksA protein / T13748, in which the compound is shown in green, and the N-terminus to C-terminus of DksA protein are marked in blue to red; B, the interaction pattern of the compound and important amino acids, the compound binds to the pocket2 region, in which amino acids R125 and L134 form hydrogen bond interactions with the compound. C, the 2D pattern of the complex interaction and the amino acids around the compound, which are mainly distributed with positively charged amino acids; D, the 2D structure of the compound, and its affinity score with the AF model is -6.94kcal / mol.

[0095] Figure 8 The interaction pattern of compound No. 6T6822 and DksA protein: A, the complex structure of DksA protein / T6822, where the compound is shown in green, and the N-terminus to C-terminus of DksA protein are marked in blue to red; B, the interaction pattern of the compound and important amino acids, the compound binds to the pocket2 region, where amino acids L95 and A128 form hydrogen bond interactions with the compound. C, the 2D pattern of the complex interaction and the amino acids around the compound, which are mainly distributed with positively charged amino acids; D, the 2D structure of the compound, and its affinity score with the AF model is -7.43kcal / mol.

[0096] Fig. 9The interaction pattern of compound No. 7T7449 and DksA protein: A, the complex structure of DksA protein / T7449, where the compound is shown in green, and the N-terminus to C-terminus of DksA protein are marked in blue to red; B, the interaction pattern of the compound and important amino acids, the compound binds to the pocket2 region, where amino acids K98 and R129 form hydrogen bond interactions with the compound. C, the 2D pattern of the complex interaction and the amino acids around the compound, which are mainly distributed with positively charged amino acids; D, the 2D structure of the compound, and its affinity score with the AF model is -5.57kcal / mol.

[0097] Fig.10 The interaction pattern of compound No. 8T7845 and DksA protein: A, the complex structure of DksA protein / T7845, in which the compound is shown in green, and the N-terminus to C-terminus of DksA protein are marked in blue to red; B, the interaction pattern of the compound and important amino acids, the compound binds to the pocket2 region, in which amino acid K98 and amino acid R129 form hydrogen bond interactions with the compound. C, the 2D pattern of the complex interaction and the amino acids around the compound, which are mainly distributed with positively charged amino acids; D, the 2D structure of the compound, and its affinity score with the AF model is -6.31kcal / mol.

[0098] Fig.11 The interaction pattern of compound No. 10T8854 and DksA protein: A, the complex structure of DksA protein / T8854, in which the compound is shown in green, and the N-terminus to C-terminus of DksA protein are marked in blue to red; B, the interaction pattern of the compound and important amino acids, the compound binds to the pocket2 region, in which amino acid K98, amino acid R129, E143 form hydrogen bond interactions with the compound. C, the 2D pattern of the complex interaction and the amino acids around the compound, which are mainly distributed with positively charged amino acids; D, the 2D structure of the compound, and its affinity score with the AF model is -6.22kcal / mol.

[0099] Fig.12The interaction pattern of compound No. 12T7691 and DksA protein: A, the complex structure of DksA protein / T7691, where the compound is shown in green, and the N-terminus to C-terminus of DksA protein are marked in blue to red; B, the interaction pattern of the compound and important amino acids, the compound binds to the pocket2 region, where amino acids K98 and R129 form hydrogen bond interactions with the compound. C, the 2D pattern of the complex interaction and the amino acids around the compound, which are mainly distributed with positively charged amino acids; D, the 2D structure of the compound, and its affinity score with the AF model is -6.11kcal / mol.

[0100] Fig.13 The interaction pattern of compound No. 19T4434 and DksA protein: A, the complex structure of DksA protein / T4434, in which the compound is shown in green, and the N-terminus to C-terminus of DksA protein are marked in blue to red; B, the interaction pattern of the compound and important amino acids, the compound binds to the pocket2 region, in which amino acids K98 and R129 form hydrogen bond interactions with the compound. C, the 2D pattern of the complex interaction and the amino acids around the compound, which are mainly distributed with positively charged amino acids; D, the 2D structure of the compound, and its affinity score with the AF model is -6.63kcal / mol.

[0101] Fig.14 The interaction pattern of compound No. 25T2525 and DksA protein: A, the complex structure of DksA protein / T2525, in which the compound is shown in green, and the N-terminus to C-terminus of DksA protein are marked in blue to red; B, the interaction pattern of the compound and important amino acids, the compound binds to the pocket2 region, in which amino acids K98 and R129 form hydrogen bond interactions with the compound. C, the 2D pattern of the complex interaction and the amino acids around the compound, which are mainly distributed with positively charged amino acids; D, the 2D structure of the compound, and its affinity score with the AF model is -5.52kcal / mol.

[0102] Fig.15The interaction pattern of compound No. 40T9285 and DksA protein: A, the complex structure of DksA protein / T9285, in which the compound is shown in green, and the N-terminus to C-terminus of DksA protein are marked in blue to red; B, the interaction pattern of the compound and important amino acids, the compound binds to the pocket2 region, and the amino acid K98 and the amino acid R129 form hydrogen bond interactions with the compound. C, the 2D pattern of the complex interaction and the amino acids around the compound, which are mainly distributed with positively charged amino acids; D, the 2D structure of the compound, and its affinity score with the AF model is -5.19kcal / mol.

[0103] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. Application of a small molecule inhibitor targeting DksA in the preparation of drugs for the treatment of Escherichia coli and Salmonella simultaneously; The small molecule inhibitor is: The amino acid sequence of DksA is shown in SEQ ID NO.1.

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